Methods and reagents for treatment and diagnosis of age-related macular degeneration

ABSTRACT

The invention relates to Factor H gene polymorphisms and haplotypes associated with an elevated or a reduced risk of AMD. The invention provides methods and reagents for diagnosis and treatment of AMD.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application Nos.60/650,078 (filed Feb. 14, 2005), 60/717,861 (filed Sep. 16, 2005),60/715,503 (filed Sep. 9, 2005), and 60/735,697 (filed Nov. 9, 2005),the entire contents of which are incorporated herein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Work described in this application has been supported, in part, by NIHEye Institute grant EY11515. The U.S. Government may have certain rightsin the invention.

BACKGROUND OF THE INVENTION

Age-related macular degeneration (AMD) is the leading cause ofirreversible vision loss in the developed world (for reviews see Zarbin,1998, 2004; Klein et al., 2004; Ambati et al., 2003; de Jong, 2004; vanLeeuwen et al., 2003) affecting approximately 15% of individuals overthe age of 60. An estimated 600 million individuals are in this agedemographic. The prevalence of AMD increases with age; mild, or earlyforms occur in nearly 30%, and advanced forms in about 7%, of thepopulation that is 75 years and older (Klein et al., 1992; Vingerling etal., 1995a, 1995b). Clinically, AMD is characterized by a progressiveloss of central vision attributable to degenerative changes that occurin the macula, a specialized region of the neural retina and underlyingtissues. In the most severe, or exudative, form of the diseaseneovascular fronds derived from the choroidal vasculature breach Bruch'smembrane and the retinal pigment epithelium (RPE) typically leading todetachment and subsequent degeneration of the retina.

AMD, a late-onset complex disorder, appears to be caused and/ormodulated by a combination of genetic and environmental factors (Seddonand Chen, 2004; Tuo et al., 2004; Klein and Francis, 2003). Familialaggregation studies have estimated the genetic component to be primarilyinvolved in as much as 25% of the disorder (Klayer et al., 1998a).According to the prevailing hypothesis, the majority of AMD cases is nota collection of multiple single-gene disorders, but instead represents aquantitative phenotype, an expression of interaction of multiplesusceptibility loci. The number of loci involved, the attributable riskconferred, and the interactions between various loci remain obscure.

Linkage and candidate gene screening analyses have provided limitedinsight into the genetics of AMD. Reliable association of one gene withincreased risk, ABCA4 (Allikmets et al., 1997) and one gene withdecreased risk, ApoE4 (Klaver et al., 1998b, Souied et al., 1998) forAMD have been reported. In addition, several groups have reportedresults of genome-wide linkage analyses (reviewed in Tuo et al., 2004;Weeks et al., 2004). Linkage of one family with AMD phenotype to aspecific chromosomal region, 1q25-q31 (ARMD1) has been documented (Kleinet al., 1998). HEMICENTIN-1 has been suggested to be the causal gene(Schultz et al., 2003) although its role has not been reliablyconfirmed. The identification of overlapping loci on chromosome 1q inseveral studies (Weeks et al., 2001; Iyengar et al., 2003; Weeks et al.,2004) suggests that this locus may harbor AMD-associated gene(s).

Recent studies of drusen, the hallmark ocular lesions associated withthe onset of AMD, have implicated a role for inflammation and otherimmune-mediated processes, in particular complement activation, in theetiology of early and late forms of AMD (Hageman et al., 1999, 2001;Mullins et al., 2000, 2001; Russell et al., 2000; Anderson et al., 2002,2004; Johnson et al., 2000, 2001; Crabb et al., 2002; Ambati et al.,2003; Penfold et al., 2001; Espinosa-Heidman et al., 2003). Thesestudies have revealed the terminal pathway complement components (C5,C6, C7, C8 and C9) and activation-specific complement protein fragmentsof the terminal pathway (C3b, iC3b, C3dg and C5b-9) as well as variouscomplement pathway regulators and inhibitors (including Factor H, FactorI, Factor D, CD55 and CD59) within drusen, along Bruch's membrane (anextracellular layer comprised of elastin and collagen that separates theRPE and the choroid) and within RPE cells overlying drusen (Johnson etal., 2000, 2001; Mullins et al. 2000, 2001; Crabb et al., 2002). Many ofthese drusen-associated molecules are circulating plasma proteinspreviously thought to be synthesized primarily by the liver.Interestingly, many also appear to be synthesized locally by RPE and/orchoroidal cells.

Activation of the complement system plays a key role in normal hostdefense and in the response to injury (Kinoshita, 1991). Inappropriateactivation and/or control of this system, often caused by mutations inspecific complement-associated genes, can contribute to autoimmunesequelae and local tissue destruction (Holers, 2003; Liszewski andAtkinson, 1991; Morgan and Walport, 1991; Shen and Meri, 2003), as hasbeen shown in atherosclerosis (Torzewski et al., 1997; Niculescu et al.,1999), Alzheimer's disease (Akiyama et al., 2000) and glomerulonephritis(Schwertz et al., 2001).

Membranoproliferative glomerulonephritis type 2 (MPGN II) is a raredisease that is associated with uncontrolled systemic activation of thealternative pathway of the complement cascade. The disease ischaracterized by the deposition of abnormal electron-dense materialcomprised of C3 and C3c, proteins involved in the alternative pathway ofcomplement, within the renal glomerular basement membrane, whicheventually leads to renal failure. Interestingly, many patients withMPGNII develop macular drusen, RPE detachments and choroidal neovascularmembranes that are clinically and compositionally indistinguishable fromthose that form in AMD, although they are often detected in the seconddecade of life (Mullins et al., 2001; O'Brien et al., 1993; Huang etal., 2003; Colville et al., 2003; Duvall-Young et al., 1989a, 1989b;Raines et al., 1989; Leys et al., 1990; McAvoy and Silvestri, 2004;Bennett et al., 1989; Orth and Ritz, 1998; Habib et al., 1975).

In most patients with MPGNII, the inability to regulate the complementcascade is mediated by an autoantibody directed against C3bBb. OtherMPGN II patients, however, harbor mutations in Factor H (Ault et al.,1997; Dragon-Durey et al., 2004) a major inhibitor of the alternativecomplement pathway. A point mutation in Factor H (1166R) causes MPGNIIin the Yorkshire pig (Jansen et al., 1998) and Factor H deficient micedevelop severe glomerulonephritis (Pickering et al., 2002). Moreover,affected individuals within some extended families with MPGNIII, arelated disorder, show linkage to chromosome 1q31-32 (Neary et al.,2002) a region that overlaps a locus that has been identified ingenome-wide linkage studies for AMD (see above). This particular locuscontains a number of complement pathway-associated genes. One group ofthese genes, referred to as the regulators of complement activation(RCA) gene cluster, contains the genes that encode Factor H, five FactorH-related genes (CFHR1, CFHR2, CFHR3, CFHR4 and CFHR5), and the betasubunit of coagulation factor XIII. A second cluster of complementpathway-associated genes, including C4BPA, C4BPB, C4BPAL2, DAF (CD55)CR1, CR2, CR1L and MCP (CD46) lies immediately adjacent to the 1q25-31locus.

BRIEF SUMMARY OF THE INVENTION

The invention relates to polymorphisms and haplotypes in the complementFactor H gene that are associated with development of age-relatedmacular degeneration (AMD) and membranoproliferative glomerulonephritistype 2 (MPGNII). The invention also relates to polymorphisms andhaplotypes in the complement Factor H-related 5 (CFHR5) genes that areassociated with development of AMD and MPGNII. The invention providesmethods of diagnosing, monitoring, and treating these and otherdiseases.

In one aspect, the invention provides a diagnostic method fordetermining a subject's propensity to develop age-related maculardegeneration (AMD), comprising detecting the presence or absence of avariation or variations at polymorphic site or polymorphic sites of theFactor H gene. In one embodiment, the invention provides methods ofdiagnosing an increased susceptibility to developing AMD involvingdetecting the presence or absence of a polymorphism in the Factor H geneof an individual. The methods may include obtaining the DNA from anindividual and analyzing the DNA from the individual to determinewhether the DNA contains a polymorphism in the Factor H gene. Certainpolymorphisms indicate the individual has an increased susceptibility todeveloping AMD relative to a control population. Certain polymorphismsindicate the individual has a reduced likelihood of developing AMD.Certain polymorphisms indicate the individual has neither an increasednor a reduced likelihood of developing AMD.

In one embodiment, a method of diagnosing a propensity to developage-related macular degeneration (AMD) in a subject involves obtaining asample of DNA from the subject and detecting in the DNA of the patientthe presence or absence of a polymorphism associated with development ofAMD, the presence of the polymorphism being an indication that thesubject has an increased propensity to develop AMD and the absence ofthe polymorphism being an indication that the subject has a reducedpropensity to develop AMD.

In a related aspect, the invention provides methods of diagnosingsusceptibility to developing AMD involving determining an individual'sFactor H haplotype. The methods include obtaining the DNA from anindividual and analyzing the DNA of the individual to determine theirFactor H haplotype. Certain haplotypes (risk haplotypes) indicate theindividual has an increased susceptibility to develop AMD. Certainhaplotypes (protective haplotypes) indicate the individual has adecreased susceptibility to develop AMD. Certain haplotypes (neutralhaplotypes) indicate the individual has neither an increased nor areduced likelihood of developing AMD.

In a related embodiment the presence or absence of a variation at apolymorphic site of the Factor H gene is determined by analysis of agene product, such as an RNA or a Factor H protein (e.g., proteinisoform) encoded by the gene. Expression of a variant protein is anindication of a variation in the Factor H gene and can indicate anincreased or reduced propensity to develop AMD. Proteins can be detectedusing immunoassays and other methods.

In another related aspect, the invention provides methods of diagnosingsusceptibility to developing AMD or other diseases by detecting avariant Factor H polypeptide in a biological sample of an individual. Inone embodiment, an antibody-based assay is used to diagnose AMD or otherdiseases in an individual by contacting a biological sample, e.g., aserum sample, of the individual with the antibody and detecting thepresence or absence of the variant Factor H polypeptide. In anembodiment, the antibody specifically interacts with an epitope specificto a variant Factor H polypeptide (i.e., not found in the wild-typeFactor H polypeptide). In an embodiment, a separation-based assay (e.g.,PAGE) is used to diagnose AMD or other diseases in an individual bydetecting the presence or absence of the variant Factor H polypeptide ina biological sample, e.g., a serum sample, of the individual.

In one aspect, the invention provides methods of treating an individualwith AMD (e.g., an individual in whom a polymorphism or haplotypeindicative of elevated risk of developing symptomatic AMD is detected)or other disease involving a variant Factor H gene by modulating thetype and/or amount of systemic and/or ocular levels of Factor H. TheFactor H polypeptide may be a wild-type Factor H polypeptide or avariant Factor H polypeptide. The Factor H polypeptide may be a Factor Hpolypeptide with a sequence encoded by neutral or protective allelesrather than alleles associated with a risk haplotype. In one embodiment,the method includes administering to the individual a Factor Hpolypeptide in an amount effective to reduce a symptom of the disease.In one embodiment, the method includes administering to an individual aFactor H polypeptide in an amount effective to reduce the propensity todevelop symptoms of the disease and delay development or progression ofthe disease. In one embodiment, the method includes administering bloodthat contains Factor H. In one embodiment, the methods includeadministering a nucleic acid (e.g., transgene) including a nucleotidesequence encoding a Factor H polypeptide. In one embodiment, the methodsinclude administering cells that express a Factor H polypeptide.

In one aspect, the invention provides methods of treating an individualwith AMD (e.g., an individual in whom a polymorphism or haplotypeindicative of elevated risk of developing symptomatic AMD is detected)or other disease involving a variant Factor H gene. In one embodiment,the method includes administering to the patient an agent that decreasesthe amount of a variant Factor H or expression of a gene encoding FactorH in an amount effective to reduce a symptom of the disease in thepatient. In a related embodiment a therapeutic amount of an inhibitor(e.g., inactivator) of the variant Factor H polypeptide in theindividual is administered.

In one embodiment an inhibitory nucleic acid (e.g., an RNA complementaryto at least a portion of the nucleotide sequence of the variant Factor Hpolypeptide) in the individual is administered. In one embodiment,purified anti-sense RNA complementary to RNA encoding a variant Factor Hpolypeptide is administered.

In another embodiment a therapeutic amount of an anti-CFH antibodysufficient to partially inactivate the variant Factor H polypeptide inthe individual is administered.

In another embodiment, the individual is treated to remove deleteriousforms of Factor H from blood (e.g., by plasmaphoresis, antibody-directedplasmaphoresis, or complexing with a Factor H binding moiety, e.g.,heparin).

In one aspect, the invention provides purified DNA encoding a variantFactor H polypeptide, purified RNA encoding a variant Factor Hpolypeptide, purified anti-sense RNA complementary to the RNA encoding avariant Factor H polypeptide, and purified variant Factor H polypeptide.In a related aspect, the invention provides nucleic acids for expressingwild-type or variant Factor H polypeptides or biologically activefragments of Factor H.

In one aspect, the invention provides gene therapy vectors comprisingnucleic acid encoding the Factor H polypeptide. The vector may include apromoter that drives expression of the Factor H gene in multiple celltypes. Alternatively, the vector may include a promoter that drivesexpression of the Factor H gene only in specific cell types, forexample, in cells of the retina or in cells of the kidney. In an aspect,pharmaceutical compositions are provided containing a gene therapyvector encoding a Factor H protein and a pharmaceutically acceptableexcipient, where the composition is free of pathogens and suitable foradministration to a human patient. In one embodiment the encoded FactorH polypeptide is a protective variant.

In one aspect, the invention provides a composition containingrecombinant or purified Factor H polypeptide, where the polypeptide is aprotective variant.

In a related aspect, the invention provides a pharmaceutical compositioncontaining recombinant or purified Factor H polypeptide and apharmaceutically acceptable excipient, where the composition is free ofpathogens and suitable for administration to a human patient. In oneembodiment the encoded Factor H polypeptide has the wild-type sequence.In one embodiment the encoded Factor H polypeptide is a protectivevariant.

In one aspect, the invention provides antibodies that specificallyinteract with a variant Factor H polypeptide but not with a wild-typeFactor H polypeptide. These antibodies may be polyclonal or monoclonaland may be obtained by subtractive techniques. These antibodies may besufficient to inactivate a variant Factor H polypeptide. In a relatedaspect, the invention provides pharmaceutical compositions containing ananti-Factor H antibody and a pharmaceutically acceptable excipient,where the composition is free of pathogens and suitable foradministration to a human patient.

In one aspect, the invention provides methods for identifying variantFactor H proteins associated with increased or reduced risk ofdeveloping AMD. In one embodiment, the invention provides a method ofidentifying a protective Factor H protein by (a) identifying anindividual as having a protective haplotype and (b) determining theamino acid sequence(s) of Factor H encoded in the genome of theindividual, where a protective Factor H protein is encoded by an allelehaving a protective haplotype. In one embodiment, the invention providesa method of identifying a neutral Factor H protein by (a) identifying anindividual as having a neutral haplotype and (b) determining the aminoacid sequence(s) of Factor H encoded in the genome of the individual,where a neutral Factor H protein is encoded by an allele having aneutral haplotype. In a related embodiment, the invention provides amethod of identifying a variant form of Factor H associated withdecreased risk of developing AMD comprising (a) identifying anindividual as having a haplotype or diplotype associated with adecreased risk of developing AMD; (b) obtaining genomic DNA or RNA fromthe individual; and (c) determining the amino acid sequence(s) of theFactor H encoded in the individual's genome, where a protective Factor Hprotein is encoded by an allele having a haplotype associated with adecreased risk of developing AMD. In an embodiment, the protective orneutral Factor H proteins do not have the amino acid sequence of thewild-type Factor H polypeptide.

In a related method, a form of Factor H associated with increased riskof developing AMD is identified by (a) identifying an individual ashaving a risk haplotype and (b) determining the amino acid sequence(s)of Factor H encoded in the genome of the individual, where a risk FactorH protein is encoded by an allele having a risk haplotype. In a relatedembodiment, the invention provides as method of identifying a variantform of Factor H associated with increased risk of developing AMDcomprising (a) identifying an individual as having a haplotype ordiplotype associated with an increased risk of developing AMD; (b)obtaining genomic DNA or RNA from the individual; and (c) determiningthe amino acid sequence(s) of the Factor H encoded in the individual'sgenome, where a risk Factor H protein is encoded by an allele having ahaplotype associated with an increased risk of developing AMD. In anembodiment, the risk Factor H proteins do not have the amino acidsequence of the wild-type Factor H polypeptide.

In one aspect, the invention provides methods of diagnosing a propensityor susceptibility to develop AMD or other diseases by detecting theratio of full-length Factor H to truncated Factor H in a biologicalsample of a patient. In one embodiment, a method of diagnosing apropensity or susceptibility to develop AMD in a subject involvesobtaining a sample of RNA from the subject and detecting in the RNA ofthe patient the ratio of expression of exon 10 (i.e., full-length FactorH) to exon 10A (i.e., truncated Factor H), the increase in ratio beingan indication that the subject has an increased propensity orsusceptibility to develop AMD and the decrease in ratio being anindication that the subject has a reduced propensity or susceptibilityto develop AMD. In one embodiment, a method of diagnosing a propensityor susceptibility to develop AMD in a subject involves obtaining asample of protein from the subject and detecting in the protein of thepatient the ratio of expression of full-length Factor H to truncatedFactor H, the increase in ratio being an indication that the subject hasan increased propensity or susceptibility to develop AMD and thedecrease in ratio being an indication that the subject has a reducedpropensity or susceptibility to develop AMD.

In one aspect, the invention provides cells containing recombinant orpurified nucleic acid encoding a Factor H protein or fragment thereof,e.g., a nucleic acid derived from the Factor H gene. The cells may bebacterial or yeast, or any other cell useful for research and drugdevelopment. Thus, the invention provides an isolated host cell or cellline expressing a recombinant variant human Factor H. In an embodiment,the variant is a risk variant and has a histidine at amino acid position402. In an embodiment, the variant is a protective variant and hasisoleucine at amino acid position 62. In an embodiment, the variant is aneutral variant. In an embodiment, the risk, protective or neutralvariant Factor H proteins do not have the amino acid sequence of thewild-type Factor H polypeptide.

In one aspect, the invention provides transgenic non-human animals whosesomatic and germ cells contain a transgene encoding a human variantFactor H polypeptide. Transgenic animals of the invention are used asmodels for AMD and for screening for agents useful in treating AMD. Theanimal may be a mouse, a worm, or any other animal useful for researchand drug development (such as recombinant production of Factor H). In anembodiment, the Factor H is a variant human Factor H, wherein saidvariant has isoleucine amino acid 62 or has histidine at amino acid 402.

In one aspect, the invention provides methods of screening forpolymorphic sites linked to polymorphic sites in the Factor H genedescribed in TABLES 1A, 1B and 1C. These methods involve identifying apolymorphic site in a gene that is linked to a polymorphic site in theFactor H gene, wherein the polymorphic form of the polymorphic site inthe Factor H gene is associated AMD, and determining haplotypes in apopulation of individuals to indicate whether the linked polymorphicsite has a polymorphic form in equilibrium disequilibrium with thepolymorphic form of the Factor H gene that associates with the AMDphenotype.

In one aspect, the invention provides diagnostic, therapeutic andscreening methods for MPGNII, carried out as described above for AMD.

In one aspect, the invention provides a diagnostic method fordetermining a subject's propensity to develop AMD or MPGNII, comprisingdetecting the presence or absence of a variation or variations atpolymorphic site or polymorphic sites of the CFHR5 gene. In oneembodiment, the invention provides methods of diagnosing an increasedsusceptibility to developing AMD or MPGNII involving detecting thepresence or absence of a polymorphism in the CFHR5 gene of anindividual. The methods may include obtaining the DNA from an individualand analyzing the DNA from the individual to determine whether the DNAcontains a polymorphism in the CFHR5 gene. Certain polymorphismsindicate the individual has an increased susceptibility to developingAMD or MPGNII. Certain polymorphisms indicate the individual has areduced likelihood of developing AMD or MPGNII. Certain polymorphismsindicate the individual has neither an increased nor a reducedlikelihood of developing AMD or MPGNII.

In one embodiment, a method of diagnosing a propensity to develop AMD orMPGNII in a subject involves obtaining a sample of DNA from the subjectand detecting in the DNA of the patient the presence or absence of apolymorphism associated with development of AMD or MPGNII, the presenceof the polymorphism being an indication that the subject has anincreased propensity to develop AMD or MPGNII and the absence of thepolymorphism being an indication that the subject has a reducedpropensity to develop AMD or MPGNII.

In a related embodiment the presence or absence of a variation at apolymorphic site of the CFHR5 gene is determined by analysis of a geneproduct, such as an RNA or a CFHR5 protein (e.g., protein isoform)encoded by the gene. Expression of a variant protein is an indication ofa variation in the CFHR5 gene and can indicate an increased or reducedpropensity to develop AMD or MPGNII. Proteins can be detected usingimmunoassays and other methods.

In a related, aspect, the invention provides methods of diagnosingsusceptibility to developing AMD or MPGNII involving determining anindividual's CFHR5 haplotype. The methods include obtaining the DNA froman individual and analyzing the DNA of the individual to determine theirCFHR5 haplotype. Certain haplotypes (risk haplotypes) indicate theindividual has an increased susceptibility to develop AMD or MPGNIIrelative to a control population. Certain haplotypes (protectivehaplotypes) indicate the individual has an decreased susceptibility todevelop AMD or MPGNII. Certain haplotypes (neutral haplotypes) indicatethe individual has neither an increased nor a reduced likelihood ofdeveloping AMD or MPGNII.

In another related, aspect, the invention provides methods of diagnosingsusceptibility to developing AMD or MPGNII or other diseases bydetecting a variant CFHR5 polypeptide in a biological sample of anindividual. In one embodiment, an antibody-based assay is used todiagnose AMD or MPGNII or other diseases in an individual by contactinga biological sample, e.g., a serum sample, of the individual with theantibody and detecting the presence or absence of the variant CFHR5polypeptide. In an embodiment, the antibody specifically interacts withan epitope specific to a variant CFHR5 polypeptide (i.e., not found inthe wild-type CFHR5 polypeptide). In an embodiment, a separation-basedassay (e.g., PAGE) is used to diagnose MPGNII or other diseases in anindividual by detecting the presence or absence of the variant CFHR5polypeptide in a biological sample, e.g., a serum sample, of theindividual. Various types of immunoassay formats can be used to assayCFH or CFHR5 polypeptide or protein in a sample. These include sandwichELISA, radioimmunoassay, fluoroimmunoassay, immunohistochemistry assay,dot-blot, dip-stick and Western Blot.

In one aspect, the invention provides methods of treating an individualwith or at risk for AMD or MPGNII (e.g., an individual in whom apolymorphism or haplotype indicative of elevated risk of developingsymptomatic AMD or MPGNII is detected) or other disease involving avariant CFHR5 gene by modulating the type and/or amount of systemicand/or renal levels of CFHR5. The CFHR5 polypeptide may be a CFHR5polypeptide encoded by neutral or protective alleles rather than allelesassociated with a risk haplotype. In one embodiment, the method includesadministering to the individual a CFHR5 polypeptide in an amounteffective to reduce a symptom of the disease. In one embodiment, themethod includes administering to an individual a CFHR5 polypeptide in anamount effective to reduce the propensity to develop symptoms of thedisease and delay development or progression of the disease. In oneembodiment, the method includes administering blood, which containsCFHR5. In one embodiment, the methods include administering a nucleicacid (e.g., transgene) including a nucleotide sequence encoding a CFHR5polypeptide.

In one aspect, the invention provides methods of treating an individualwith AMD or MPGNII (e.g., an individual in whom a polymorphism orhaplotype indicative of elevated risk of developing symptomatic AMD orMPGNII is detected) or other disease involving a variant CFHR5 gene. Inone embodiment, the method includes administering to the patient anagent that decreases the amount of a variant CFHR5 or expression of agene encoding CFHR5 in an amount effective to reduce a symptom of thedisease in the patient. The CFHR5 polypeptide may be a wild-type CFHR5polypeptide or a variant CFHR5 polypeptide.

In one embodiment an inhibitory nucleic acid (e.g., an RNA complementaryto at least a portion of the nucleotide sequence of the variant CFHR5polypeptide) in the individual is administered. In one embodiment,purified anti-sense RNA complementary to RNA encoding a variant CFHR5polypeptide is administered.

In another embodiment a therapeutic amount of an anti-CFHR5 antibodysufficient to partially inactivate the variant CFHR5 polypeptide in theindividual is administered.

In a related embodiment a therapeutic amount of an inhibitor (e.g.,inactivator) of the variant CFHR5 polypeptide in the individual isadministered.

In another embodiment, the individual is treated to remove deleteriousforms of CFHR5 from blood (e.g., by plasmaphoresis, antibody-directedplasmaphoresis, or complexing with a CFHR5 binding moiety, e.g.,heparin).

In one aspect, the invention provides purified DNA encoding a variantCFHR5 polypeptide, purified RNA encoding a variant CFHR5 polypeptide,purified anti-sense RNA complementary to the RNA encoding a variantCFHR5 polypeptide, and purified variant CFHR5 polypeptide. In a relatedaspect, the invention provides nucleic acids for expressing wild-type orvariant CFHR5 polypeptides or biologically active fragments of CFHR5.

In one aspect, the invention provides gene therapy vectors comprisingnucleic acid encoding the CFHR5 polypeptide. The vector may include apromoter that drives expression of the CFHR5 gene in multiple celltypes. Alternatively, the vector may include a promoter that drivesexpression of the CFHR5 gene only in specific cell types, for example,in cells of the retina or cells of the kidney (e.g., endothelial cells,mesangial cells, podocytes). In an aspect, pharmaceutical compositionsare provided containing a gene therapy vector encoding a CFHR5 proteinand a pharmaceutically acceptable excipient, where the composition isfree of pathogens and suitable for administration to a human patient. Inone embodiment the encoded CFHR5 polypeptide is a protective variant.

In one aspect, the invention provides a composition containingrecombinant or purified CFHR5 polypeptide, where the polypeptide is aprotective variant.

In a related aspect, the invention provides a pharmaceutical compositioncontaining recombinant or purified CFHR5 polypeptide and apharmaceutically acceptable excipient, where the composition is free ofpathogens and suitable for administration to a human patient. In oneembodiment the encoded CFHR5 polypeptide has the wild-type sequence. Inone embodiment the encoded CFHR5 polypeptide is a protective variant.

In one aspect, the invention provides antibodies that specificallyinteract with a variant CFHR5 polypeptide but not with a wild-type CFHR5polypeptide. These antibodies may be polyclonal or monoclonal and may beobtained by subtractive techniques. These antibodies may be sufficientto inactivate a variant CFHR5 polypeptide. In a related aspect, theinvention provides pharmaceutical compositions containing an anti-CFHR5antibody and a pharmaceutically acceptable excipient, where thecomposition is free of pathogens and suitable for administration to ahuman patient.

In one aspect, the invention provides methods for identifying variantCFHR5 proteins associated with increased or reduced risk of developingAMD or MPGNII. In one embodiment, the invention provides a method ofidentifying a protective CFHR5 protein by (a) identifying an individualas having a protective haplotype and (b) determining the amino acidsequence(s) of CFHR5 encoded in the genome of the individual, where aprotective CFHR5 protein is encoded by an allele having a protectivehaplotype. In one embodiment, the invention provides a method ofidentifying a neutral CFHR5 protein by (a) identifying an individual ashaving a neutral haplotype and (b) determining the amino acidsequence(s) of CFHR5 encoded in the genome of the individual, where aneutral CFHR5 protein is encoded by an allele having a neutralhaplotype. In a related embodiment, the invention provides as method ofidentifying a variant form of CFHR5 associated with decreased risk ofdeveloping AMD or MPGNII comprising (a) identifying an individual ashaving a haplotype or diplotype associated with a decreased risk ofdeveloping AMD or MPGNII; (b) obtaining genomic DNA, or RNA, from theindividual; and (c) determining the amino acid sequence(s) of the CFHR5encoded in the individual's genome, where a protective CFHR5 protein isencoded by an allele having a haplotype associated with a decreased riskof developing AMD or MPGNII. In an embodiment, the protective or neutralCFHR5 proteins do not have the amino acid sequence of the wild-typeCFHR5 polypeptide.

In a related method, a form of CFHR5 associated with increased risk ofdeveloping AMD or MPGNII is identified by (a) identifying an individualas having a risk haplotype and (b) determining the amino acidsequence(s) of CFHR5 encoded in the genome of the individual, where arisk CFHR5 protein is encoded by an allele having a risk haplotype. In arelated embodiment, the invention provides as method of identifying avariant form of CFHR5 associated with increased risk of developing AMDor MPGNII comprising (a) identifying an individual as having a haplotypeor diplotype associated with an increased risk of developing AMD orMPGNII; (b) obtaining genomic DNA or RNA from the individual; and (c)determining the amino acid sequence(s) of the CFHR5 encoded in theindividual's genome, where a risk CFHR5 protein is encoded by an allelehaving a haplotype associated with an increased risk of developing AMDor MPGNII. In an embodiment, the risk CFHR5 proteins do not have theamino acid sequence of the wild-type CFHR5 polypeptide.

In one aspect, the invention provides cells containing recombinant orpurified nucleic acid derived from the CFHR5 gene. The cells may bebacterial or yeast, or any other cell useful for research and drugdevelopment. Thus, the invention provides an isolated host cell or cellline expressing a recombinant variant human CFHR5. In an embodiment, theCFHR5 variant is a risk variant and has a serine at amino acid position46. In an embodiment, the CFHR5 variant is a neutral variant. In anembodiment, the risk, protective or neutral variant CFHR5 proteins doesnot have the amino acid sequence of the wild-type CFHR5 polypeptide.

In one aspect, the invention provides transgenic non-human animals whosesomatic and germ cells contain a transgene encoding a human variantCFHR5 polypeptide. Transgenic animals of the invention are used asmodels for AMD or MPGNII and for screening for agents useful in treatingAMD or MPGNII. The animal may be a mouse, a worm, or any other animaluseful for research and drug development (such as recombinant productionof CFHR5). In an embodiment, the CFHR5 is a variant human CFHR5, whereinsaid CFHR5 variant has serine at amino acid 46.

In one aspect, the invention provides methods of screening forpolymorphic sites linked to polymorphic sites in the CFHR5 genedescribed in TABLE 14 or TABLE 15. These methods involve identifying apolymorphic site in a gene that is linked to a polymorphic site in theCFHR5 gene, wherein the polymorphic form of the polymorphic site in theCFHR5 gene is associated with AMD or MPGNII, and determining haplotypesin a population of individuals to indicate whether the linkedpolymorphic site has a polymorphic form in equilibrium disequilibriumwith the polymorphic form of the CFHR5 gene that associates with the AMDor MPGNII phenotype.

In one aspect, the invention provides kits for analysis of a Factor Hhaplotype. The kits may be used for diagnosis of AMD in a patient. Thekits may include one or more Factor H Factor H allele-specificoligonucleotides (e.g., allele-specific primers or probes), orantibodies that specifically recognize the Factor H polypeptide. TheFactor H allele-specific oligonucleotides may include sequences derivedfrom the coding (exons) or non-coding (promoter, 5′ untranslated,introns or 3′ untranslated) region of the Factor H gene. The FactorH-specific antibodies may recognize the normal or wild-type Hpolypeptide or variant Factor H polypeptides in which one or morenon-synonymous single nucleotide polymorphisms (SNPs) are present in theFactor H coding region. The kits may be used to diagnose AMD, as well asother diseases associated with SNPs in the Factor H gene, such asMPGNII. The kits may include instead, or in addition, one or more FactorH-Related 5 (CFHR5) allele-specific oligonucleotides (e.g., primers andprobes), or antibodies that specifically recognize the CFHR5polypeptide. The CFHR5 allele-specific primers and Factor H-related 5allele-specific oligonucleotides may include sequences derived from thecoding (exons) or non-coding (promoter, 5′ untranslated, introns or 3′untranslated) region of the Factor H-related 5 gene. The FactorH-related 5-specific antibodies may recognize the normal or wild-type Hpolypeptide or variant Factor H-related 5 polypeptides in which one ormore non-synonymous single nucleotide polymorphisms (SNPs) are presentin the Factor H-related 5 coding region.

In one embodiment the kit contains probes or primers that distinguishalleles at a polymorphic site listed in TABLE 1A, TABLE 1B and/or TABLE1C. In an embodiment the probes are primers for nucleic acidamplification of a region spanning a Factor H gene polymorphic sitelisted in TABLE 1A, TABLE 1B and/or TABLE 1C. In an embodiment the kithas probes or primers that distinguish alleles at more than onepolymorphic site listed in TABLE 1A, TABLE 1B and/or TABLE IC. In anembodiment the kit has probes or primers that distinguish alleles atmore than one polymorphic site, where the polymorphic site includes: (a)rs529825; (b) rs800292; (c) rs3766404; (d) rs1061147; (e) rs1061170; (f)rs203674; (g) at least one of rs529825 and rs800292; (h) at least one ofrs1061147, rs106170 and rs203674; (i) at least one of rs529825 andrs800292; and rs3766404; and at least one of rs1061147, rs1061170 andrs203674; or (j) at least rs529825, rs800292, rs3766404, rs1061170 andrs203674.

In a related embodiment the kit has probes or primers that distinguishalleles at more than one polymorphic site, where the polymorphic siteincludes: (a) rs529825; (b) rs800292; (c) intron 2 (IVS2 or insTT) (d)rs3766404; (e) rs1061147; (f) rs1061170; (g) exon 10A; (h) rs203674; (i)rs375046; (j) rs529825 and rs800292; (k) at least two or three ofrs1061147, rs1061170 and rs203674; (l) at least one of rs529825 andrs800292; and intron 2; and rs3766404; and at least one of rs1061147,rs1061170 and rs203674; and exon 10A; and, rs375046; (m) at leastrs529825; rs800292; intron 2; rs3766404; rs1061170; exon 10A; rs203674;and rs375046; (n) at least two, or at least three, or at least four ofrs529825, rs800292, intron 2; rs3766404, rs1061170, exon 10A, rs203674,and rs375046; (o) exon 22 (1210); or (p) exon 22 (1210) in combinationwith any aforementioned variation or set of variations (a-o). In anembodiment the kit has probes or primers that distinguish alleles at oneor both of rs460897 and rs460184. In an embodiment the kit has probes orprimers that distinguish alleles at more than one polymorphic site,where the polymorphic sites are selected from: (a) rs3753394; (b)rs529825; (c) rs800292; (d) intron 2 (IVS2 or insTT); (e) rs3766404; (f)rs1061147; (g) rs1061170; (h) rs2274700; (i) rs203674; (j) rs3753396;and (k) rs1065489.

In one embodiment the kit contains, instead of, or in addition to, theprobes described above, probes, primers, antibodies and the like thatdistinguish polymorphic sites in the CFHR5 gene. In a one aspect, theinvention provides kits for the diagnosis of AMD or MPGNII in a patientbased on variation in the CFHR5 gene. The kits may include one or moreCFHR5-specific probes or CFHR5 allele-specific oligonucleotides, orantibodies that specifically recognize the CFHR5 polypeptide. TheCFHR5-specific primers and CFHR5 allele-specific oligonucleotides mayinclude sequences derived from the coding (exons) or non-coding(promoter, 5′ untranslated, introns or 3′ untranslated) region of theCFHR5 gene. The CFHR5-specific antibodies may recognize the normal orwild-type CFHR5 polypeptide or variant CFHR5 polypeptides in which oneor more non-synonymous single nucleotide polymorphisms (SNPs) arepresent in the CFHR5 coding region. The kits may be used to diagnose AMDor MPGNII, as well as other diseases associated with SNPs in the CFHR5gene.

In one embodiment the kit contains probes or primers that distinguishalleles at a polymorphic site listed in TABLE 14 or TABLE 15. In anembodiment the probes are primers for nucleic acid amplification of aregion spanning a CFHR5 gene polymorphic site listed in TABLE 14 orTABLE 15. In an embodiment the kit has probes or primers thatdistinguish alleles at more than one polymorphic site listed in TABLE 14or TABLE 15. In an embodiment the kit comprises probes or primers thatdistinguish alleles one, two or all of the following polymorphic sites:rs9427661 (−249T>C); rs9427662 (−20T>C); and rs12097550 (P46S).

In one embodiment the kit contains probes or primers that distinguishalleles at a polymorphic site in the CFH gene and at a polymorphic sitein a CFHR gene, such as CFHR5.

In one aspect, the invention provides devices for determining asubject's haplotype. The devices are useful for, for example, thediagnosis of AMD or other diseases in a patient. In one embodiment thedevice contains probes or primers that distinguish alleles at apolymorphic site listed in TABLE 1A, 1B and/or 1C. In an embodiment theprobes are primers for nucleic acid amplification of a region spanning aFactor H gene polymorphic site listed in TABLE 1A, 1B and/or 1C. In anembodiment the device has probes or primers that distinguish alleles atmore than one polymorphic site listed in TABLE 1A, 1B and/or 1C. In anembodiment the device has probes or primers that distinguish alleles atmore than one polymorphic site, where the polymorphic site includes (a)rs529825; (b) rs800292; (c) rs3766404; (d) rs1061147; (e) rs1061170; (f)rs203674; (g) at least one of rs529825 and rs800292; (h) at least one ofrs1061147, rs1061170 and rs203674; (i) at least one of rs529825 andrs800292; and rs3766404; and at least one of rs10611147, rs10611170 andrs203674; or (j) at least rs529825, rs800292, rs3766404, rs1061170 andrs203674.

The kits described above and their contents may also be used to identitya propensity to develop MPGNII or to determine a Factor H haplotype forany purpose.

In a related embodiment the device has probes or primers thatdistinguish alleles at more than one polymorphic site, where thepolymorphic site includes: (a) rs529825; (b) rs800292; (c) intron 2(IVS2 or insTT) (d) rs3766404; (e) rs1061147; (f) rs1061170; (g) exon10A; (h) rs203674; (i) rs375046; (j) rs529825 and rs800292; (k) at leasttwo or three of rs1061147, rs1061170 and rs203674; (l) at least one ofrs529825 and rs800292; and intron 2; and rs3766404; and at least one ofrs10611147, rs1061170 and rs203674; and exon 10A; and rs375046; (m) atleast rs529825; rs800292; intron 2; rs3766404; rs1061170; exon 10A;rs203674; and rs375046; (n) at least two, or at least three, or at leastfour of rs529825; rs800292; intron 2; rs3766404; rs1061170; exon 10A;rs203674; and rs375046; (o) exon 22 (1210); or (p) exon 22 (1210) incombination with any aforementioned variation or set of variations(a-o). In an embodiment the device has probes or primers thatdistinguish alleles at one or both of rs460897 and rs460184. In anembodiment the device has probes or primers that distinguish alleles atmore than one polymorphic site, where the polymorphic sites are selectedfrom: (a) rs3753394; (b) rs529825; (c) rs800292; (d) intron 2 (IVS2 orinsTT); (e) rs3766404; (f) rs1061147; (g) rs1061170; (h) rs2274700; (i)rs203674; (j) rs3753396; and (k) rs1065489. In an embodiment the devicehas probes or primers that distinguish alleles at more than onepolymorphic site, where the polymorphic sites are selected from: (a)rs3753394; (b) rs529825; (c) rs800292; (d) intron 2 (IVS2 or insTT); (e)rs3766404; (f) rs1061147; (g) rs1061170; (h) rs2274700; (i) rs203674;(j) rs3753396; and (k) rs1065489.

In a one aspect, the invention provides devices for the diagnosis of AMDor MPGNII in a patient. In one embodiment the device contains probes orprimers that distinguish alleles at a polymorphic site listed in TABLE14 or TABLE 15. In an embodiment the probes are primers for nucleic acidamplification of a region spanning a CFHR5 gene polymorphic site listedin TABLE 14 or TABLE 15. In an embodiment the device has probes orprimers that distinguish alleles at more than one polymorphic sitelisted in TABLE 14 or TABLE 15. Devices of the invention may containprobes or primers that distinguish between both Factor H and CHFR5variants, including any combination of the sites described above andelsewhere in this disclosure.

The devices described above and their contents may also be used toidentity a propensity to develop MPGNII or to determine a Factor Hhaplotype for any purpose.

In one embodiment the device contains, instead of, or in addition to,the probes or primers described above, probes, primers, antibodies andthe like that distinguish polymorphic sites in the CFHR5 gene. In a oneaspect, the invention provides devices for the diagnosis of AMD orMPGNII in a patient based on variation in the CFHR5 gene. The devicesmay include one or more CFHR5-specific probes or CFHR5 allele-specificoligonucleotides, or antibodies that specifically recognize the CFHR5polypeptide. The CFHR5-specific primers and CFHR5 allele-specificoligonucleotides may include sequences derived from the coding (exons)or non-coding (promoter, 5′ untranslated, introns or 3′ untranslated)region of the CFHR5 gene. The CFHR5-specific antibodies may recognizethe normal or wild-type CFHR5 polypeptide or variant CFHR5 polypeptidesin which one or more non-synonymous single nucleotide polymorphisms(SNPs) are present in the CFHR5 coding region. The devices may be usedto diagnose AMD or MPGNII, as well as other diseases associated withSNPs in the CFHR5 gene.

In one embodiment the device contains probes or primers that distinguishalleles at a polymorphic site listed in TABLE 14 or TABLE 15. In anembodiment the probes are primers for nucleic acid amplification of aregion spanning a CFHR5 gene polymorphic site listed in TABLE 14 orTABLE 15. In an embodiment the device has probes or primers thatdistinguish alleles at more than one polymorphic site listed in TABLE 14or TABLE 15. In an embodiment the kit comprises probes or primers thatdistinguish alleles one, two or all of the following polymorphic sites:rs9427661 (−249T>C); rs9427662 (−20T>C); and rs12097550 (P46S).

In one embodiment the device contains probes or primers that distinguishalleles at a polymorphic site in the CFH gene and at a polymorphic sitein a CFHR gene, such as CFHR5.

Additional aspects of the invention will be apparent upon reading theentire disclosure.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1L show the immunolocalization of Factor H (FIGS. 1A-1H) andthe terminal complement complex (C5b-9) (FIGS. 1I-1L) in the humanretinal pigmented epithelium. Abbreviations: (RPE)-choroid (Chor)complex; Bruch's membrane (BM); Retina (Ret); Drusen (Dr).

FIG. 2 shows RT-PCR analysis of Factor H gene expression (CFH and thetruncated form HFL1) using RNA extracted from the human eye.

FIG. 3 is a diagram of the human Factor H gene showing the approximatelocations of 12 SNPs used in the analysis, the 22 exons of the Factor Hgene, the 20 short consensus repeats (SCRs), the binding sites forpathogens and other substrates, and the linkage disequilibrium (LD)blocks. The diagram, showing all 22 exons of CFH (but not introns) isnot drawn to scale.

FIG. 4 is a haplotype network diagram of human Factor H gene SNPsshowing the relationship between the risk (filled-in circles),protective (lined circles), neutral (open circles) and ancestral(indicated) haplotypes and the relative frequency of the haplotypes, asindicated by the sizes and positions of the circles.

FIG. 5 shows an association analysis of human Factor H gene haplotypesand diplotypes. Eight informative SNPs were analyzed for pairwiselinkage disequilibrium in AMD cases and controls. The nucleotide on thecoding strand at the indicated polymorphic sites is shown, except forIVS1, where the nucleotide on the non-coding strand is shown.

FIG. 6 shows the 3926 base nucleotide sequence of the reference form ofhuman Factor H cDNA (GenBank accession number Y00716 [SEQ ID NO:1]). TheATG initiation codon begins at nucleotide position 74 and the TAGtermination codon ends at nucleotide position 3769.

FIG. 7 shows the polypeptide sequence encoded by SEQ ID NO:1 (GenBankaccession number Y00716 [SEQ ID NO:2]). The 1231 amino acid Factor Hpolypeptide includes an 18 amino acid N-terminal signal peptide.

FIG. 8 shows the 1658 base nucleotide sequence of the reference form ofHFL1, the truncated form of the human Factor H (GenBank accession numberX07523 [SEQ ID NO:3]). The ATG initiation codon begins at nucleotideposition 74 and the TGA termination codon ends at nucleotide position1423.

FIG. 9 shows the polypeptide sequence of the reference form of HFL1encoded by SEQ ID NO:3 (GenBank accession number X07523 [SEQ ID NO:4]).The 449 amino acid HFL1 polypeptide includes an 18 amino acid N-terminalsignal peptide.

FIG. 10 shows the polypeptide sequence of an exemplary protectivevariant of human Factor H [SEQ ID NO:5]. This protective variant FactorH polypeptide has a isoleucine at amino acid position 62 and a tyrosineat amino acid position 402 (indicated in bold).

FIG. 11 shows the polypeptide sequence of an exemplary protectivevariant of HFL1, the truncated form of human Factor H (SEQ ID NO:6).This protective variant truncated Factor H polypeptide has a isoleucineat amino acid position 62 and tyrosine at amino acid position 402(indicated in bold).

FIG. 12 shows marked glomerular hypercellularity with denseintramembranous deposits that cause capillary wall thickening in apatient with MPGNII, as viewed by (A) light microscopy and (B) electronmicroscopy. The deposits can form a segmental, discontinuous or diffusepattern in the lamina densa of the glomerular basement membrane (GBM).By light microscopy, they are eosinophilic and refractile, stainbrightly with periodic acid-Schiff and are highly osmophilic, whichexplains their electron-dense appearance (A). Even by electronmicroscopy the deposits lack substructure and appear as very darkhomogeneous smudges (B). The exact composition of dense deposits remainsunknown (bar, 5 μm).

FIG. 13 is a diagram showing the activation and regulation of thealternative pathway of the complement cascade, which is systematicallyactivated at a high level in patients with AMD and MPGNII. Thealternative pathway of the complement cascade is systematicallyactivated at a high level in patients with MPGN II/DDD. Normally,continuous low-level activation of C3 occurs by a process of spontaneoushydrolysis known as tick-over. C3 hydrolysis is associated with a largeconformational protein change shown at the top of the diagram. Theconformational change makes C3(H20) similar to C3b, a C3 cleavageproduct. The initial convertase, C3(H2O)Bb, activates C3 to form C3b.Although C3b has a fleeting half-life, if it binds to IgG, cells orbasement membranes, it is protected from immediate inactivation.(C3b)2-IgG complexes form in the fluid phase and bind properdin (P),which facilitates factor B binding and the generation of C3bBb, theconvertase of the alternative pathway, shown here as aBb(C3b)2-IgG-properdin complex. The amplification loop is depicted bythe arrows. C3NeF prolongs the half-life of C3 convertase and is shownin the inset. One mechanism to degrade C3 convertase is through itsinteraction with complement Factor H(CFH), shown at the bottom right asfH. Deficiency of and mutations in Factor H are associated with MPGNII/DDD.

FIG. 14 is a diagram showing the organization of theregulators-of-complement-activation (RCA) gene cluster on chromosome1q32 and the arrangement of approximately 60-amino acid domains known asshort consensus repeats (SCRs) in complement Factor H(CFH), FactorH-Like 1 (CFHL1) and Factor H-Related 1, 2, 3, 4 and 5 (CFHR1, CFHR2,CFHR3, CFHR4 and CFHR5). CFH has 20 SCRs. The interacting partners withsome of these SCRs has been determined and is shown on the top right(CRP, C reactive protein; Hep, heparin). Complement factor H-like 1(CFHL1) is a splice isoform of CFH, while complement factor H-relatedproteins 1-5 (CFHR1-5) are each encoded by a unique gene (CFHR1-5). TheSCRs of CFHR1-5 are similar to some of the SCRs in CFH, as denoted bythe numbers in the ovals. For example, CFHR5 has 9 SCRs, with the firsttwo being similar to SCRs 6 and 7 of Factor H and therefore having CRPand heparin binding properties. SCRs5-7 of CFHR5 have the numbers 12-14within the corresponding ovals because these SCRs are similar to SCRs12-14 of Factor H and have C3b and heparin binding properties.

FIG. 15 shows a linkage disequilibrium plot indicating that A307A andY402H are in linkage disequilibrium in Factor H and −249T>C and −20T>Care in linkage disequilibrium in CFHR5.

FIG. 16 shows the 2821 base nucleotide sequence of the reference form ofhuman CFHR5 (GenBank accession number AF295327 [SEQ ID NO:7]. The ATGinitiation codon begins at nucleotide position 94 and the TGAtermination codon ends at nucleotide position 1803.

FIG. 17 shows the polypeptide sequence encoded by SEQ ID NO:7 (GenBankaccession number AAK15619 [SEQ ID NO:8]. The 569 amino acid CFHR5polypeptide includes an 18 amino acid N-terminal signal peptide.

FIG. 18 shows genomic duplications in the genes for CFH and the FactorH-related proteins. Exons are indicated as vertical lines. Regionslabeled with the same letter (e.g., A, A′, and A″) have substantiallyidentical sequences.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The invention provides a collection of polymorphisms and haplotypescomprised of multiple variations in the Factor H gene, and in FactorH-related genes such as Factor H-Related 5 gene. These polymorphisms andhaplotypes are associated with age related macular degeneration (AMD)and other Factor H-related conditions. Certain of these polymorphismsand haplotypes result in variant Factor H polypeptides. Detection ofthese and other polymorphisms and sets of polymorphisms (e.g.,haplotypes) is useful in designing and performing diagnostic assays forAMD. Polymorphisms and sets of polymorphisms can be detected by analysisof nucleic acids, by analysis of polypeptides encoded by Factor H codingsequences (including polypeptides encoded by splice variants), or byother means known in the art. Analysis of such polymorphisms andhaplotypes is also useful in designing prophylactic and therapeuticregimes for AMD.

Factor H is a multifunctional protein that functions as a key regulatorof the complement system. See Zipfel, 2001, “Factor H and disease: acomplement regulator affects vital body functions” Semin Thromb Hemost.27:191-9. The Factor H protein activities include: (1) binding toC-reactive protein (CRP), (2) binding to C3b, (3) binding to heparin,(4) binding to sialic acid; (5) binding to endothelial cell surfaces,(6) binding to cellular integrin receptors (7) binding to pathogens,including microbes (see FIG. 3), and (8) C3b co-factor activity. TheFactor H gene, known as HF1, CFH and HF, is located on human chromosome1, at position 1q32. The 1q32 particular locus contains a number ofcomplement pathway-associated genes. One group of these genes, referredto as the regulators of complement activation (RCA) gene cluster,contains the genes that encode Factor H, five Factor H-related genes(FHR-1, FHR-2, FHR-3, FHR-4 and FHR-5 or CFHR1, CFHR2, CFHR3, CFHR4 andCFHR5, respectively), and the gene encoding the beta subunit ofcoagulation factor XIII. The Factor H and Factor H related genes iscomposed almost entirely of short consensus repeats (SCRs). Factor H andFHL1 are composed of SCRs 1-20 and 1-7, respectively. FHR-1, FHR-2,FHR-3, FHR-4 and FHR-5 are composed of 5, 4, 5, 5 and 8 SCRs,respectively (see FIG. 14). The order of genes, from centromere totelomere is FH/FHL1, FHR-3, FHR-1, FHR-4, FHR-2 and FHR-5.

Factor H Gene

The reference form of human Factor H cDNA (SEQ ID NO:1) (see Ripoche etal., 1988, Biochem J 249:593-602) and genomic sequences have beendetermined. The Factor H cDNA encodes a polypeptide 1231 amino acids inlength (SEQ ID NO:2) having an apparent molecular weight of 155 kDa.There is an alternatively spliced form of Factor H is known as FHL-1(and also has been referred to as HFL1 or CFHT). FHL-1 (SEQ ID NO:3)corresponds essentially to exons 1 through 9 of Factor H (see Ripoche etal., 1988, Biochem J 249:593-602). The FHL1 cDNA encodes a polypeptide449 amino acids in length (SEQ ID NO:4) having an apparent molecularweight of 45-50 kDA. The first 445 amino acids of FH1 and FHL1 areidentical, with FHL1 having a unique C-terminal 4 amino acids (exon10A). The alternative exon 10A is located in the intron between exon 9and exon 10. cDNA and amino acid sequence data for human Factor H andFHL1 are found in the EMBL/GenBank Data Libraries under accessionnumbers Y00716 and X07523, respectively. The 3926 base nucleotidesequence of the reference form of human Factor H cDNA (GenBank accessionnumber Y00716 [SEQ ID NO:1]) is shown in FIG. 6, and the polypeptidesequence encoded by SEQ ID NO:1 (GenBank accession number Y00716 [SEQ IDNO:2]) is shown in FIG. 7. The 1658 base nucleotide sequence of thereference form of HFL1, the truncated form of the human Factor H(GenBank accession number X07523 [SEQ ID NO:3]) is shown in FIG. 8, andthe polypeptide sequence encoded by SEQ ID NO:3 (GenBank accessionnumber X07523 [SEQ ID NO:4]) is shown in FIG. 9. The Factor H genesequence (150626 bases in length) is found under GenBank accessionnumber AL049744. The Factor H promoter is located 5′ to the codingregion of the Factor H gene.

FHR-1 Gene

The FHR-1 gene is also known as CHFR1, CFHL1, CFHL, FHR1 and HFL1. Thereference form of human HFR-1 cDNA (see Estaller et al., 1991, J.Immunol. 146:3190-3196) and genomic sequences have been determined. TheFHR-1 cDNA encodes a polypeptide 330 amino acids in length having anpredicted molecular weight of 39 kDa. cDNA and amino acid sequence datafor human FHR-1 are found in the EMBL/GenBank Data Libraries underaccession number M65292. The FHR-1 gene sequence is found under GenBankaccession number AL049741.

FHR-2 Gene

The FHR-2 gene is also known as CHFR2, CFHL2, FHR2 and HFL3. Thereference form of human HFR-2 cDNA (see Strausberg et al., Proc. Natl.Acad. Sci. USA 99:16899-16903) and genomic sequences have beendetermined. The FHR-2 cDNA encodes a polypeptide 270 amino acids inlength having a predicted molecular weight of 31 kDa. cDNA and aminoacid sequence data for human FHR-2 are found in the EMBL/GenBank DataLibraries under accession number BC022283. The FHR-2 gene sequence isfound under GenBank accession number AL139418.

FHR-3 Gene

The FHR-3 gene is also known as CFHR3, CFHL3, FHR3 and HLF4. Thereference form of human HFR-3 cDNA (see Strausberg et al., Proc. Natl.Acad. Sci. USA 99:16899-16903) and genomic sequences have beendetermined. The FHR-3 cDNA encodes a polypeptide 330 amino acids inlength having a predicted molecular weight of 38 kDa. cDNA and aminoacid sequence data for human FHR-3 are found in the EMBL/GenBank DataLibraries under accession number BC058009. The FHR-3 gene sequence isfound under GenBank accession number AL049741.

FHR-4 Gene

The FHR-4 gene is also known as CFHR4, CFHL4 and FHR4. The referenceform of human HFR-4 cDNA (see Skerka et al., 1991, J. Biol. Chem.272:5627-5634) and genomic sequences have been determined. The FHR-4cDNA encodes a polypeptide 331 amino acids in length having a predictedmolecular weight of 38 kDa. cDNA and amino acid sequence data for humanFHR-4 are found in the EMBL/GenBank Data Libraries under accessionnumber X98337. The FHR-4 gene sequence is found under GenBank accessionnumbers AF190816 (5′ end), AL139418 (3′ end) and BX248415.

FHR-5 Gene

The FHR-5 gene is also known as CFHR5, CFHL5 and FHR5. The referenceform of human CFHR5 cDNA (SEQ ID NO:83) (see McRae et al., 2001, J.Biol. Chem. 276:6747-6754) and genomic sequences have been determined.The CFHR5 cDNA encodes a polypeptide 569 amino acids in length (SEQ IDNO:8) having an apparent molecular weight of 65 kDa. cDNA and amino acidsequence data for human CFHR5 are found in the EMBL/GenBank DataLibraries under accession number AF295327. The 2821 base nucleotidesequence of the reference form of human CFHR5 (GenBank accession numberAF295327 [SEQ ID NO:7] is shown in FIG. 16, and the polypeptide sequenceencoded by SEQ ID NO:7 (GenBank accession number AAK15619 [SEQ ID NO:8]is shown in FIG. 17. The CFHR5 gene sequence is found under GenBankaccession numbers AL139418 (5′ end) and AL353809 (3′ end). The FHR-5promoter is located 5′ to the coding region of the CFHR5 gene.

II. Definitions

The following definitions are provided to aid in understanding theinvention. Unless otherwise defined, all terms of art, notations andother scientific or medical terms or terminology used herein areintended to have the meanings commonly understood by those of skill inthe arts of medicine and molecular biology. In some cases, terms withcommonly understood meanings are defined herein for clarity and/or forready reference, and the inclusion of such definitions herein should notbe assumed to represent a substantial difference over what is generallyunderstood in the art.

A “nucleic acid”, “polynucleotide” or “oligonucleotide” is a polymericform of nucleotides of any length, may be DNA or RNA, and may be single-or double-stranded. Nucleic acids may include promoters or otherregulatory sequences. Oligonucleotides are usually prepared by syntheticmeans. Nucleic acids include segments of DNA, or their complementsspanning or flanking any one of the polymorphic sites shown in TABLE 1A,TABLE 1B and/or TABLE 1C or otherwise known in the Factor H gene. Thesegments are usually between 5 and 100 contiguous bases, and often rangefrom a lower limit of 5, 10, 12, 15, 20, or 25 nucleotides to an upperlimit of 10, 15, 20, 25, 30, 50 or 100 nucleotides (where the upperlimit is greater than the lower limit). Nucleic acids between 5-10,5-20, 10-20, 12-30, 15-30, 10-50, 20-50 or 20-100 bases are common. Thepolymorphic site can occur within any position of the segment. Areference to the sequence of one strand of a double-stranded nucleicacid defines the complementary sequence and except where otherwise clearfrom context, a reference to one strand of a nucleic acid also refers toits complement. For certain applications, nucleic acid (e.g., RNA)molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theuse of phosphorothioate or 2′-O-methyl rather than phosphodiesteraselinkages within the backbone of the molecule. Modified nucleic acidsinclude peptide nucleic acids (PNAs) and nucleic acids withnontraditional bases such as inosine, queosine and wybutosine andacetyl-, methyl-, thio- and similarly modified forms of adenine,cytidine, guanine, thymine, and uridine which are not as easilyrecognized by endogenous endonucleases.

“Hybridization probes” are nucleic acids capable of binding in abase-specific manner to a complementary strand of nucleic acid. Suchprobes include nucleic acids and peptide nucleic acids (Nielsen et al.,1991). Hybridization may be performed under stringent conditions whichare known in the art. For example, see, e.g., Berger and Kimmel (1987)Methods In Enzymology, Vol. 152: Guide To Molecular Cloning Techniques,San Diego: Academic Press, Inc.; Sambrook et al. (1989) MolecularCloning: A Laboratory Manual, 2nd Ed., Vols. 1-3, Cold Spring HarborLaboratory; Sambook (2001) 3rd Edition; Rychlik, W. and Rhoads, R. E.,1989, Nucl. Acids Res. 17, 8543; Mueller, P. R. et al. (1993) In:Current Protocols in Molecular Biology 15.5, Greene PublishingAssociates, Inc. and John Wiley and Sons, New York; and Anderson andYoung, Quantitative Filter Hybridization in Nucleic Acid Hybridization(1985)). As used herein, the term “probe” includes primers. Probes andprimers are sometimes referred to as “oligonucleotides.”

The term “primer” refers to a single-stranded oligonucleotide capable ofacting as a point of initiation of template-directed DNA synthesis underappropriate conditions, in an appropriate buffer and at a suitabletemperature. The appropriate length of a primer depends on the intendeduse of the primer but typically ranges from 15 to 30 nucleotides. Aprimer sequence need not be exactly complementary to a template but mustbe sufficiently complementary to hybridize with a template. The term“primer site” refers to the area of the target DNA to which a primerhybridizes. The term “primer pair” means a set of primers including a 5′upstream primer, which hybridizes to the 5′ end of the DNA sequence tobe amplified and a 3′ downstream primer, which hybridizes to thecomplement of the 3′ end of the sequence to be amplified.

Exemplary hybridization conditions for short probes and primers is about5 to 12 degrees C. below the calculated Tm. Formulas for calculating Tmare known and include: Tm=4° C.×(number of G's and C's in the primer)+2°C.×(number of A's and T's in the primer) for oligos <14 bases andassumes a reaction is carried out in the presence of 50 mM monovalentcations. For longer oligos, the following formula can be used: Tm=64.9°C.+41° C.×(number of G's and C's in the primer−16.4)/N, where N is thelength of the primer. Another commonly used formula takes into accountthe salt concentration of the reaction (Rychlik, supra, Sambrook, supra,Mueller, supra.): Tm=81.5° C.+16.6° C.×(log 10[Na+]+[K+])+0.41° C.×(%GC)−675/N, where N is the number of nucleotides in the oligo. Theaforementioned formulae provide a starting point for certainapplications; however, the design of particular probes and primers maytake into account additional or different factors. Methods for design ofprobes and primers for use in the methods of the invention are wellknown in the art.

The terms “risk,” “protective,” and “neutral” are used to describevariations, SNPS, haplotypes, diplotypes, and proteins in a populationencoded by genes characterized by such patterns of variations. A riskhaplotype is an allelic form of a gene, herein Factor H or a FactorH-related gene, comprising at least one variant polymorphism, andpreferably a set of variant polymorphisms, associated with increasedrisk for developing AMD. The term “variant” when used in reference to aFactor H or Factor H-related gene, refers to a nucleotide sequence inwhich the sequence differs from the sequence most prevalent in apopulation, herein humans of European-American descent. The variantpolymorphisms can be in the coding or non-coding portions of the gene.An example of a risk Factor H haplotype is the allele of the Factor Hgene encoding histidine at amino acid 402 and/or cysteine at amino acid1210. The risk haplotype can be naturally occurring or can besynthesized by recombinant techniques. A protective haplotype is anallelic form of a gene, herein Factor H or a Factor H-related gene,comprising at least one variant polymorphism, and preferably a set ofvariant polymorphisms, associated with decreased risk of developing AMD.For example, one protective Factor H haplotype has an allele of theFactor H gene encoding isoleucine at amino acid 62. The protectivehaplotype can be naturally occurring or synthesized by recombinanttechniques. A neutral haplotype is an allelic form of a gene, hereinFactor H or a Factor H-related gene, that does not contain a variantpolymorphism associated in a population or ethnic group with eitherincreased or decreased risk of developing AMD. It will be clear from thefollowing discussion that a protein encoded in a “neutral” haplotype maybe protective when administered to a patient in need of treatment orprophylaxis for AMD or other conditions. That is, both “neutral” and“protective” forms of CFH or CFHR5 can provide therapeutic benefit whenadministered to, for example, a subject with AMD or risk for developingAMD, and thus can “protect” the subject from disease.

The term “wild-type” refers to a nucleic acid or polypeptide in whichthe sequence is a form prevalent in a population, herein humans ofEuropean-American descent (approximately 40% prevalence; see FIG. 5).For purposes of this disclosure, a “wild-type” Factor H protein has thesequence of SEQ ID NO:2 (FIG. 7), except that the amino acid at position402 is tyrosine (Y; [SEQ ID NO:337]). For purposes of this disclosure, aFactor H gene encoding a wild-type Factor H protein has the sequence ofSEQ ID NO:1 (FIG. 6), except that the codon beginning at base 1277,corresponding to the amino acid at position 402 encodes tyrosine (TAT[SEQ ID NO:336]).

The term “variant” when used in reference to a Factor H or FactorH-related polypeptide, refers to a polypeptide in which the sequencediffers from the normal or wild-type sequence at a position that changesthe amino acid sequence of the encoded polypeptide. For example, somevariations or substitutions in the nucleotide sequence of Factor H genealter a codon so that a different amino acid is encoded (for example andnot for limitation, having an alternative allele at one or more of 162V,Y402H, D936E) resulting in a variant polypeptide. Variant polypeptidescan be associated with risk (e.g., having histidine at position 402),associated with protection (e.g., having isoleucine at position 62), orcan be encoded by a neutral haplotype (e.g., having aspartic acid atposition 936). Variant CFHR5 polypeptides can be associated with risk(e.g., having serine at position 46), associated with protection, or canbe neutral.

The term “reference” when referring to a Factor H polypeptide means apolypeptide in which the amino acid sequence is identical to thesequence described by Ripoche et al., 1988, Biochem J. 249:593-602) forfull-length (FH1, SEQ ID NO:2) or truncated (FHL1, SEQ ID NO:4) humanFactor H. The term “reference” when referring to a CFHR5 polypeptidemeans a polypeptide in which the amino acid sequence is identical to thesequence described by McRae et al., 2001, J. Biol. Chem. 276:6747-6754)for full-length human CFHR5 (SEQ ID NO:8). The first identified allelicform is arbitrarily designated the reference form or allele; otherallelic forms are designated as alternative or variant alleles.Wild-type and variant forms may have substantial sequence identity withthe reference form (e.g., the wild-type or variant form may be identicalto the reference form at least 90% of the amino acid positions of thewild-type or variant, sometimes at least 95% of the positions andsometimes at least 98% or 99% of the positions). A variant may differfrom a reference form in certain regions of the protein due to aframeshift mutation or splice variation.

The term “polymorphism” refers to the occurrence of two or moregenetically determined alternative sequences or alleles in a population.A “polymorphic site” is the locus at which sequence divergence occurs.Polymorphic sites have at least two alleles. A diallelic polymorphismhas two alleles. A triallelic polymorphism has three alleles. Diploidorganisms may be homozygous or heterozygous for allelic forms. Apolymorphic site may be as small as one base pair. Examples ofpolymorphic sites include: restriction fragment length polymorphisms(RFLPs); variable number of tandem repeats (VNTRs); hypervariableregions; minisatellites; dinucleotide repeats; trinucleotide repeats;tetranucleotide repeats; and simple sequence repeats. As used herein,reference to a “polymorphism” can encompass a set of polymorphisms(i.e., a haplotype).

A “single nucleotide polymorphism (SNP)” occurs at a polymorphic siteoccupied by a single nucleotide, which is the site of variation betweenallelic sequences. The site is usually preceded by and followed byhighly conserved sequences of the allele. A SNP usually arises due tosubstitution of one nucleotide for another at the polymorphic site.Replacement of one purine by another purine or one pyrimidine by anotherpyrimidine is called a transition. Replacement of a purine by apyrimidine or vice versa is called a transversion. A synonymous SNPrefers to a substitution of one nucleotide for another in the codingregion that does not change the amino acid sequence of the encodedpolypeptide. A non-synonymous SNP refers to a substitution of onenucleotide for another in the coding region that changes the amino acidsequence of the encoded polypeptide. A SNP may also arise from adeletion or an insertion of a nucleotide or nucleotides relative to areference allele.

A “set” of polymorphisms means more than one polymorphism, e.g., atleast 2, at least 3, at least 4, at least 5, at least 6, or more than 6of the polymorphisms shown in TABLE 1A, TABLE 1B and/or TABLE 1C orotherwise known in the Factor H gene or other gene.

The term “haplotype” refers to the designation of a set of polymorphismsor alleles of polymorphic sites within a gene of an individual. Forexample, a “112” Factor H haplotype refers to the Factor H genecomprising allele 1 at each of the first two polymorphic sites andallele 2 at the third polymorphic site. A “diplotype” is a haplotypepair.

An “isolated” nucleic acid means a nucleic acid species that is thepredominant species present in a composition. Isolated means the nucleicacid is separated from at least one compound with which it is associatedin nature. A purified nucleic acid comprises (on a molar basis) at leastabout 50, 80 or 90 percent of all macromolecular species present.

Two amino acid sequences are considered to have “substantial identity”when they are at least about 80% identical, preferably at least about90% identical, more preferably at least about 95%, at least about 98%identical or at least about 99% identical. Percentage sequence identityis typically calculated by determining the optimal alignment between twosequences and comparing the two sequences. Optimal alignment ofsequences may be conducted by inspection, or using the local homologyalgorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2: 482, usingthe homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol.Biol. 48: 443, using the search for similarity method of Pearson andLipman, 1988, Proc. Natl. Acad. Sci. U.S.A. 85: 2444, by computerizedimplementations of these algorithms (e.g., in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.) using default parameters for amino acid comparisons (e.g., forgap-scoring, etc.). It is sometimes desirable to describe sequenceidentity between two sequences in reference to a particular length orregion (e.g., two sequences may be described as having at least 95%identity over a length of at least 500 basepairs). Usually the lengthwill be at least about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900or 1000 amino acids, or the full length of the reference protein. Twoamino acid sequences can also be considered to have substantial identityif they differ by 1, 2, or 3 residues, or by from 2-20 residues, 2-10residues, 3-20 residues, or 3-10 residues.

“Linkage” describes the tendency of genes, alleles, loci or geneticmarkers to be inherited together as a result of their location on thesame chromosome. Linkage can be measured by percent recombinationbetween the two genes, alleles, loci or genetic markers. Typically, locioccurring within a 50 centimorgan (cM) distance of each other arelinked. Linked markers may occur within the same gene or gene cluster.“Linkage disequilibrium” or “allelic association” means the preferentialassociation of a particular allele or genetic marker with a specificallele or genetic marker at a nearby chromosomal location morefrequently than expected by chance for any particular allele frequencyin the population. A marker in linkage disequilibrium can beparticularly useful in detecting susceptibility to disease, even if themarker itself does not cause the disease.

The terms “diagnose” and “diagnosis” refer to the ability to determinewhether an individual has the propensity to develop disease (includingwith or without signs or symptoms). Diagnosis of propensity to developdisease can also be called “screening” and, as used herein, the termsdiagnosis and screening are used interchangeably. It will be appreciatedthat having an increased or decreased propensity to developing acondition refers to the likelihood of developing the condition relativeto individuals in the population without the condition.

III. Tables

Certain tables referred to herein are provided following the Examples,infra. The following descriptions are provided to assist the reader:

TABLES 1A-1C show human Factor H gene polymorphisms and theirassociation with age-related macular degeneration. (1A) The dbSNP no.,location, sequences of the coding (top, 5′ to 3′ direction) andnon-coding (bottom) strands spanning the polymorphisms, amino acidchanges, allele frequencies for the control and AMD cases, and χ² andP-values for 1 SNPs in the human Factor H gene are shown. (1B) The dbSNPno., interrogated sequences, corresponding nucleotide in the chimpFactor H gene, location, amino acid changes, and sets of primers andprobes for 11 SNPs in the human Factor H gene are shown. (1C) Thelocation, sequences spanning the polymorphisms, amino acid position andamino acid change, if any, for 14 SNPs in the human Factor H gene thatare not found in the dbSNP database are shown.

TABLE 2 shows a haplotype analysis of eight SNPs in the human Factor Hgene in a cohort of AMD cases and controls.

TABLE 3 shows a haplotype analysis of six SNPs in the human Factor Hgene and their association with AMD.

TABLE 4 shows the association of 11 human Factor H gene SNPs withage-related macular degeneration.

TABLE 5 shows the primers used for SSCP, DHPLC and DNA sequencinganalysis for human Factor H.

TABLE 6 shows genotyping data of AMD patients and controls.

TABLE 7 shows the frequency of an at-risk haplotype in various ethnicgroups.

TABLE 8 shows several Factor H diplotypes. Common risk and protectivediplotypes are indicated.

TABLE 9 shows the sequences of primers used to amplify the Factor Hcoding sequence.

TABLE 10 shows the sequences of primers used to amplify the CFHR5 codingsequence.

TABLE 11 shows an analysis of Factor H SNPs in 22 MPGNII patients.

TABLE 12 shows a comparison of Factor H SNP frequencies in 22 MPGNIIpatients and AMD-negative, ethnically matched controls.

TABLE 13 lists Factor H SNPs associated with MPGNII and their relatedSCR.

TABLE 14 shows an analysis of CFHR5 SNPs in 22 MPGNII patients.

TABLE 15 shows a comparison of CFHR5 SNP frequencies in 22 MPGNIIpatients and AMD-negative, ethnically matched controls.

TABLE 16 shows exemplary allele-specific probes (16A) and primers (16B)useful for detecting polymorphisms in the Factor H gene.

IV. Complement Factor H Polymorphisms

In one aspect, the invention provides new diagnostic, treatment and drugscreening methods related to the discovery that polymorphic sites in theComplement Factor H(HF1) gene are associated with susceptibility to anddevelopment of AMD.

Factor H polymorphisms associated with AMD were identified as describedin Example 1, by examining the coding and adjacent intronic regions ofFactor H (including exon 10A, which is transcribed for the Factor Hisoform FHL1) for variants using SSCP analysis, DHPLC analysis, anddirect sequencing, according to standard protocols. Remainingpolymorphisms were typed by the 5′ nuclease (Taqman, ABI) methodology.Taqman genotyping and association analysis were performed as described(Gold et al., 2004). Primers for SSCP and DNA sequencing analyses weredesigned to amplify each exon and its adjacent intronic regions usingMac Vector software. PCR-derived amplicons were screened for sequencevariation by SSCP and DHPLC according to standard protocols. All changesdetected by SSCP and DHPLC were confirmed by bidirectional sequencingaccording to standard protocols. Statistical analyses were performedusing chi-square (χ²) and Fisher's exact tests (P values).

Two independent groups of AMD cases and age-matched controls were used.All participating individuals were of European-American descent, overthe age of 60 and enrolled under IRB-approved protocols followinginformed consent. These groups were comprised of 352 unrelated patientswith clinically documented AMD (mean age 79.5±7.8 years) and 113unrelated, control patients (mean age 78.4±7.4 years; matched by age andethnicity) from the University of Iowa, and 550 unrelated patients withclinically documented AMD (mean age 71.32±8.9 years) and 275 unrelated,matched by age and ethnicity, controls (mean age 68.84±8.6 years) fromColumbia University. Patients were examined by indirect opthalmoscopyand slit-lamp microscopy by retina fellowship-trained ophthalmologists.

Fundas photographs were graded according to a standardized,international classification system (Bird et al., 1995). Controlpatients were selected and included if they did not exhibit anydistinguishing signs of macular disease or have a known family historyof AMD. The AMD patients were subdivided into phenotypic categories:early AMD (ARM), geographic atrophy (GA), and exudative (CNV) AMD, basedupon the classification of their most severe eye at the time of theirentry into the study. The University of Iowa ARM and GA cases werefurther subdivided into distinct phenotypes (RPE changes alone, >10macular hard drusen, macular soft drusen, BB (cuticular) drusen, PED,“Cherokee” atrophy, peninsular geographic atrophy and pattern geographicatrophy). The earliest documentable phenotype for all cases was alsorecorded and employed in the analyses.

As shown in TABLE 1A, a highly significant association of polymorphicsites in the Factor H gene with AMD was found in an examination of twoindependent cohorts that together included approximately 900 AMD casesand 400 matched controls. Sixteen (16) polymorphisms in the Factor Hgene are listed in TABLES 1A-1B. Of these twelve (12) are found in theSNP database (dbSNP) which may be found in the National Center forBiotechnology Information (NCBI). The dbSNP is a collection of SNPs inthe human Factor H gene which are dispersed among the 22 coding exons ofthe Factor H gene and among the promoter, the 5′ untranslated region,the introns, and the 3′ untranslated region of the Factor H gene. Listedbelow are the accession numbers for 379 SNPs in the human Factor H genethat are found in the dbSNP database. These SNPs can be used in carryingout methods of the invention.

TABLE A rs17575212 rs11582939 rs7551203 rs5014736 rs2019724 rs534479rs395963 rs17573867 rs11580821 rs7546015 rs5014735 rs1984894 rs534399rs395544 rs16840522 rs11579439 rs7540032 rs5014734 rs1928433 rs529825rs395129 rs16840465 rs11539862 rs7539005 rs5014733 rs1928432 rs528298rs393955 rs16840462 rs11398897 rs7537967 rs5003626 rs1887973 rs521605rs386258 rs16840422 rs11390840 rs7535653 rs5003625 rs1831282 rs520992rs385892 rs16840419 rs11340441 rs7535263 rs5003624 rs1831281 rs519839rs385543 rs16840410 rs11339120 rs7529589 rs5002880 rs1831280r rs518957rs383191 rs16840401 rs11318544 rs7526622 rs5002876 rs1410997 rs551397rs405306 rs16840397 rs11285593 rs7524776 rs5002875 rs1410996 rs544889rs403846 rs16840394 rs10922109 rs7522681 rs5002874 rs1329429 rs543879rs402991 rs16840381 rs10922108 rs7519439 rs4658046 rs1329428 rs536564rs402056 rs16840379 rs10922107 rs7514261 rs4657826 rs1329427 rs536539rs399469 rs12756364 rs10922106 rs7513157 rs4350148 rs1329424 rs515299rs398248 rs12746361 rs10922105 rs7415913 rs4044888 rs1329423 rs514756rs381974 rs12740961 rs10922104 rs7413999 rs4044884 rs1329422 rs514591rs380390 rs12726401 rs10922103 rs7413137 rs4044882 rs1329421 rs513699rs380060 rs12566629 rs10922102 rs6695321 rs3834020 rs1299282 rs512900rs379489 rs12565418 rs10922101 rs6691749 rs3766405 rs1292487 rs508505rs375046 rs12406047 rs10922100 rs6690982 rs3766404 rs1292477 rs499807rs374896 rs12405238 rs10922099 rs6689826 rs3766403 rs1292476 rs495968rs374231 rs12402808 rs10922098 rs6689009 rs3753397 rs1292475 rs495222rs371647 rs12238983 rs10922097 rs6688272 rs3753396 rs1292474 rs493367rs368465 rs12144939 rs10922096 rs6685249 rs3753395 rs1292473 rs491480rs364947 rs12136675 rs10922095 rs6682138 rs3753394 rs1292472 rs490864rs203688 rs12134975 rs10922094 rs6680396 rs3043115 rs1292471 rs488738rs203687 rs12134598 rs10922093 rs6677604 rs3043113 rs1292466 rs487114rs203686 rs12127759 rs10922092 rs6677460 rs3043112 rs1156679 rs482934rs203685 rs12124794 rs10801561 rs6677089 rs3043111 rs1156678 rs480266rs203684 rs12116702 rs10801560 rs6675088 rs2878649 rs1089031 rs466287rs203683r rs12096637 rs10801559 rs6674960 rs2878648 rs1065489 rs464798s203682 rs12085209 rs10801558 rs6673106 rs2878647 rs1061171 rs463726rs203681 rs12081550 rs10801557 rs6664877 rs2860102 rs1061170 rs460897rs203680 rs12069060 rs10801556 rs6664705 rs2746965 rs1061147 rs460787rs203679 rs12047565 rs10801555 rs6660100 rs2336225 rs1061111 rs460184rs203678 rs12047106 rs10801554 rs6428357 rs2336224 rs1060821 rs459598rs203677 rs12047103 rs10801553 rs6428356 rs2336223 rs1048663 rs454652rs203676 rs12045503 rs10754200 rs5779848 rs2336222 rs1040597 rs436337rs203675 rs12042805 rs10754199 rs5779847 rs2336221 rs800295 rs435628rs203674 rs12041668 rs10737680 rs5779846 rs2300430 rs800293 rs434536rs203673 rs12040718 rs10737679 rs5779845 rs2300429 rs800292 rs430173rs203672 rs12039905 rs10733086 rs5779844 rs2284664 rs800291 rs428060rs203671 rs12038674 rs10688557 rs5022901 rs2284663 rs800290 rs424535rs203670 rs12038333 rs10685027 rs5022900 rs2274700 rs800280 rs422851rs203669 rs12033127 rs10664537 rs5022899 rs2268343 rs800271 rs422404rs70621 rs12032372 rs10616982 rs5022898 rs2173383 rs800269 rs420922rs70620 rs12030500 rs10545544 rs5022897 rs2143912 rs766001 rs420921rs15809 rs12029785 rs10540668 rs5016801 rs2104714 rs765774 rs419137rs14473 rs12025861 rs10536523 rs5014740 rs2064456 rs742855 rs414539rs3645 rs11809183 rs10489456 rs5014739 rs2020130 rs731557 rs412852rs11801630 rs10465603 rs5014738 rs2019727 rs572515 rs410232 rs11799956rs10465586 rs5014737 rs1803696 rs570618 rs409953 rs11799595 rs9970784rs5002879 rs1587325 rs569219 rs409319 rs11799380 rs9970075 rs5002878rs1576340 rs564657 rs409308 rs11584505 rs9427909 rs5002877 rs1474792rs559350 rs407361

Two frequent non-synonymous variants, 162V in exon 2 and Y420H in exon9, and a less frequent variant, R1210c in exon 22, exhibited the mostsignificant association with AMD.

Three additional polymorphisms in TABLES 1A-1B are not found in the SNPdatabase: a polymorphism in the promoter (promoter 1 in TABLE 1A); apolymorphism in intron 2 in which two T nucleotides are inserted; and apolymorphism in Exon 10A.

The first column in TABLE 1A lists the dbSNP number for polymorphisms inthe Factor H gene. For example, rs800292 is the dbSNP designation for apolymorphism in the Factor H gene. A description of this polymorphism,as well as the other Factor H gene polymorphisms in dbSNP, is availableat http://www.ncbi.nlm.nih.gov(http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=snp&cmd=search&term=).The second column lists the location of the polymorphism. For example,the rs800292 polymorphism is located in exon 2 of the Factor H gene.Polymorphisms not identified by a database number can be referred to bylocation (e.g., “intron 2”). The third column lists the nucleic acidsequence of the coding (top, 5′ to 3′ direction) and non-coding (bottom)strands of DNA spanning the polymorphisms. For example, the rs800292polymorphism, G or A as indicated in the brackets for the coding strand,is flanked by the 20 nucleotides shown 5′ and 3′ to the polymorphism.“N” in the sequence spanning the Exon 10A polymorphism indicates theinsertion of a single nucleotide, either A, C, G or T, in the variantallele. The fourth column lists the SEQ ID NO: for the sequences. Thefifth column lists the amino acid change, if any, associated with thepolymorphism. For example, the rs800292 polymorphism results in a changein the amino acid sequence from valine (V) to isoleucine (I) at position62 of the Factor H polypeptide. The sixth column lists the allelefrequency of the polymorphism in a control population. The numbers 1 and2 refer to the alleles that correspond to the first and secondnucleotide, respectively, at the polymorphic site in the third column.For example, for the rs800292 polymorphism, G is present in 78% and A ispresent in 22% of the alleles sequenced from the control population. Theseventh column lists the allele frequency of the polymorphism in an AMDpopulation. For example, for the rs800292 polymorphism, G is present in91% and A is present in 9% of the alleles sequenced from an AMDpopulation. The eighth column lists the chi-square and Fisher's exacttests (χ² and P values, respectively) for the comparison between theallele frequencies of the polymorphism in the control and AMDpopulations. For example, for the rs800292 polymorphism, the χ² value is16.19 and the P value is 5.74×10⁻⁵, indicating that the G allele isassociated with AMD.

The first column in TABLE 1B parts (1), (2) and (3) lists the dbSNPnumber for polymorphisms in the Factor H gene. For part (1), the secondcolumn lists the nucleic acid sequence spanning the polymorphisms(interrogated sequence). For the rs529825 (intron 1), rs800292 (exon 2),and rs203674 (intron 10) polymorphisms, the sequences of the non-codingstrand of the human Factor H gene are shown. The third column lists theSEQ ID NOs: for the sequences. The fourth column lists the allelepresent in the chimp Factor H gene. The fifth column lists the locationof the SNP. The sixth column lists the amino acid change, if any,associated with the polymorphism. For part (2), the second and fourthcolumns list the forward and reverse primers or AOD numbers foramplifying the polymorphisms. The third and fifth columns list the SEQID NOs: for the primers. For part (3), the second and fourth columnslist probes used for detecting the polymorphisms. The third and fifthcolumns list the SEQ ID NOs: for the probes.

It should be understood that additional polymorphic sites in the FactorH gene, which are not listed in TABLES 1A-1B may be associated with AMD.Exemplary polymorphic sites in the Factor H gene are listed, for exampleand not limitation, above. TABLE 1C lists an additional 14 polymorphicsites in the Factor H gene, which are not found in the dbSNP database,that may be associated with AMD or other diseases. The first columnlists the location of the SNP. The second column lists the nucleic acidsequence spanning the polymorphisms. “notG” in the sequence spanning theExon 5 polymorphism indicates the presence of an A, C or T nucleotide inthe variant allele. “not C” in the sequences spanning the Exon 6polymorphisms indicates the presence of an A, G or T nucleotide in thevariant allele. “N” in the sequences spanning the Exon 21 polymorphismindicates the insertion of a single nucleotide, either A, C, G or T, inthe variant allele. The third column lists the amino acid change, if anyassociated with the polymorphism. The fourth column lists the SEQ IDNOs: for the sequences. These SNPs can also be used in carrying outmethods of the invention. Moreover, it will be appreciated that theseCFH polymorphisms are useful for linkage and association studies,genotyping clinical populations, correlation of genotype information tophenotype information, loss of heterozygosity analysis, andidentification of the source of a cell sample.

TABLE 2 shows a haplotype analysis of eight SNPs in the human Factor Hgene in AMD cases and controls. The at-risk haplotypes are shown instippled boxes, with the haplotype determining SNPs (Y402H and IVS10)shown in denser stippling. The protective haplotypes are shown indiagonal-lined boxes, with the haplotype determining SNPs (IVS1, 162Vand IVS6) shown indenser diagonal lines. The first column lists theallele of the polymorphism in the promoter (Prom). The second columnlists the allele of the non-coding strand of the polymorphism in intron1 (IVS1). The third column lists the allele of the non-coding strand ofthe polymorphism in exon 2 (162V). The fourth column lists the allele ofthe polymorphism in intron 6 (IVS6). The fifth column lists the alleleof the polymorphism in Exon 9 (Y402H). The sixth column lists the alleleof the non-coding strand of the polymorphism in intron 10 (IVS10). Theseventh column lists the allele of the polymorphism in Exon 13 (Q672Q).The eighth column lists the allele of the polymorphism in Exon 18(D936E). The dbSNP designations for these eight SNPs are listed inTABLES 1A-1B. The ninth column lists the Odds Ratio (OR) for thehaplotype. The tenth column lists the P value for the at-risk and twoprotective haplotypes. The eleventh and twelfth columns list thefrequencies of the haploptype in AMD cases and controls.

TABLE 3 shows a haplotype analysis of six Factor H polymorphisms withAMD. The first column lists certain alleles of the polymorphism in thepromoter (rs3753394). The second column lists the allele of thepolymorphism in intron 1 (rs529825). The third column lists the alleleof the polymorphism in intron 6 (rs3766404). The fourth column lists thepolymorphism in intron 10 (rs203674). The fifth column lists the alleleof the polymorphism in exon 13 (rs3753396). The sixth column lists theallele of the polymorphism in exon 18 (rs1065489). The numbers 1 and 2in columns 1 to 6 refer to the alleles that correspond to the first andsecond nucleotide, respectively, at each of the polymorphic sites (seeTABLE 1A). Thus, columns 1 to 6 list the alleles of polymorphisms from5′ to 3′ in the Factor H gene. The seventh column lists the Factor Hhaplotype based on the polymorphisms listed in columns 1 to 6. Theeighth column lists the frequency of the indicated Factor H haplotype ina control population. The ninth column lists the frequency of theindicated Factor H haplotype in the AMD population. As shown in TABLE 3,the haplotype analysis suggests that multiple variants contribute to theassociation and may confer either elevated or reduced risk of AMD.

TABLE 8 shows a diplotype analysis of seven Factor H polymorphisms. Thefirst column indicates whether the diplotype is associated withincreased (risk diplotype) or decreased (protective diplotype) risk ofdeveloping AMD. Common risk and protective diplotypes are indicated. Thesecond column lists the alleles of the polymorphism in exon 2 (162V).The third column lists the alleles of the polymorphism in intron 2(IVS2-18). The fourth column lists the alleles of the polymorphism inexon 9 (Y402H). The fifth column lists the alleles of the polymorphismin exon 18 (D936E). The sixth column lists the alleles of thepolymorphism in intron 20 (IVS20).

Risk-Associated (“Risk”) Polymorphisms and Haplotypes

Sites comprising polymorphisms associated with increased risk for AMDare shown in TABLE 1A and TABLE 2. Polymorphisms particularly associatedwith increased risk include a variant allele at: rs1061170 (402H; exon9); rs203674 (intron 10) and the polymorphism at residue 1210 (R1210C;exon 22).

Certain haplotypes associated with increased risk for AMD are shown inTABLES 2 and 6 and FIG. 5. As shown in TABLE 2 and FIG. 5, one commonat-risk haplotype is the H1 haplotype, which includes the variant alleleat position 402 (encoding histidine) and the variant allele at IVS10(intron 10, rs203674) and is found in 49% of AMD cases, but only in 26%of controls. Homozygotes for the risk diplotype (H1/H1) aresignificantly at risk. Other at-risk haplotypes and diplotypes are shownin TABLES 2 and 8. Similar data are presented in TABLE 3, which shows anat-risk haplotype (111211) found in 48% of AMD cases, but only in 28% ofcontrols.

Notably, seventy percent of MPGN II (membranoproliferativeglomerulonephritis type II) patients harbor this at-risk haplotype (seeTABLE 7), indicating that propensity to develop MPGNII can be detectedand treated as described herein for AMD.

Significant associations of these polymorphic sites were also found withvarious AMD subtypes, as disclosed in Example 1.

The non-synonymous polymorphism at amino acid position 1210 in exon 22of the Factor H gene is strongly associated with AMD (see TABLE 1A). Thevariant allele, which encodes a cysteine instead of an arginine, isfound in the heterozygous state in 5% of AMD cases, and no controls in acohort comprised of 919 individuals ascertained at the University ofIowa. No 1210C homozygotes have been identified to date. The presence ofcysteine at amino acid position 1210 of Factor H, therefore, provides astrong indication that the individual has AMD or is likely to developAMD. Remarkably, 1210C is indicative of propensity to develop AMD orother complement mediated conditions even when detected on allele thatis otherwise protective (e.g., Y402). Variation at CFH position 1210(R1210C) is known to cause atypical hemolytic uremic syndrome (aHUS), acomplement related disease with renal manifestations. By extension,other CFH variations or mutations known to cause aHUS may be associatedwith an increased risk for developing AMD. The most common establishedaHUS-causing variations include, but are not limited to, T956M, Q1076E,D1119G, W1183L, T1184R, L1189R, L1189F, S1191W, S1191L, V1197A, andR1215G (Esparza-Gordillo et al 2005; Perez-Caballero et al 2001;Richards et al 2001; Sanchez-Corral et al 2002); additional aHUS-causingmutations are described in Saunders (Saunders et al 2006). In one aspectof the present invention, a biological sample from a subject (e.g.,protein or nucleic acid) is assayed for the presence of one or moreaHUS-associated variations or mutations, the presence of which isindicative of a propensity to develop AMD.

It will be appreciated that additional polymorphic sites in the Factor Hgene, which are not listed in TABLES 1A-1C, may further refine thishaplotype analysis. A haplotype analysis using non-synonymouspolymorphisms in the Factor H gene is useful to identify variant FactorH polypeptides. Other haplotypes associated with risk may encode aprotein with the same sequence as a protein encoded by a neutral orprotective haplotype, but contain an allele in a promoter or intron, forexample, that changes the level or site of Factor H expression. It willalso be appreciated that a polymorphism in the Factor H gene, or in aFactor H-related gene, may be linked to a variation in a neighboringgene. The variation in the neighboring gene may result in a change inexpression or form of an encoded protein and have detrimental orprotective effects in the carrier.

Protective Polymorphisms and Haplotypes

Unexpectedly, protective polymorphisms and haplotypes were alsodiscovered. For example, as shown in TABLE 2 and FIG. 5, the protectiveH2 haplotype, including a variant allele in IVS6 (intron 6, rs3766404)occurs in 12% of controls, but only in 6% of AMD cases. The protectiveH4 haplotype includes the variant allele in IVS1 (intron 1, rs529825)and the variant allele (162) (exon 2, rs800292) and occurs in 18% ofcontrols, but only in 12% of AMD cases. Similar data is presented inTABLE 3, where the haplotype 121111 occurs in 21% of controls, but onlyin 13% of AMD cases and the haploptype 112111 occurs in 13% of controls,but only in 6% of AMD cases. As shown in FIG. 5, homozygotes with aprotective haplotype are significantly protected.

In some cases the protein encoded by a gene characterized by aprotective haplotype has a sequence different from risk haplotypeproteins (e.g., due to the presence of a nonsynomous SNP). For example,a protective form of Factor H protein generally does not have histidineat position 402. In some embodiments a protective form has isoleucine atposition 62. Additional protective forms can be identified by (1)identifying an individual or individuals with a protective haplotype and(2) determining the sequence(s) of Factor H cDNA or protein from theindividuals. Other protective forms are identified as described below inSection VIII.

Neutral Polymorphisms and Haplotypes

Certain haplotypes are associated in a population with neither increasedrisk nor decreased risk of developing AMD and are referred to as“neutral.” Examples of neutral haplotypes identified in a Caucasianpopulation are shown in FIG. 5 (H3 and H5). Additional or differentneutral haplotypes may be identified in racially/ethnically differentpopulations. Proteins encoded by a gene characterized by a neutralhaplotype are “neutral” Factor H proteins. As explained supra, “neutral”Factor H proteins could provide therapeutic benefit when administered topatients having a risk haplotype or diagnosed with AMD. For example,exemplary proteins encoded by genes characterized by a neutral haplotypeinclude proteins not having histidine at position 402 and/or not havingisoleucine at position 62. A protein not having histidine at position402 may have tyrosine at that position, or may have an amino acid otherthan histidine or tyrosine. A protein not having isoleucine at position62 may have valine at that position, or may have an amino acid otherthan valine or isoleucine. A neutral form of Factor H protein generallydoes not have cysteine at position 1210.

V. Factor H Related 5 (CFHR5) Gene Polymorphisms

In one aspect, the invention provides new diagnostic, treatment and drugscreening methods related to the discovery that polymorphic sites in theFactor H and CFHR5 genes are associated with susceptibility to anddevelopment of MPGNII.

Factor H and CFHR5 polymorphisms associated with MPGNII were identifiedas described in Example 2, by examining the coding and adjacent intronicregions of Factor H or CFHR5 for variants using PCR amplification,followed by agarose gel electrophoresis and bi-directional sequencingaccording to standard protocols to verify PCR products. Novel andreported SNPs were typed in the control population by denaturing highperformance liquid chromatography (DHPLC). Primers used to amplify theFactor H and CFHR5 coding sequences are shown in TABLES 9 and 10,respectively.

The test group consisted of patients with biopsy-proven MPGNII wereascertained in nephrology divisions and enrolled in this study underIRB-approved guidelines. The control group consisted ofethnically-matched, but not age-matched, unrelated persons in whom AMDhad been excluded by opthalmologic examination.

As shown in TABLES 11 and 12, a significant association of polymorphicsites in the Factor H gene with MPGNII was found in an examination of 22MPGNII cases and 131 ethnically-matched controls. Eleven (11)polymorphisms in the Factor H gene are listed in TABLES 11 and 12. Ofthese, six (6) are found in the SNP database (dbSNP) which may be foundin the National Center for Biotechnology Information (NCBI). The dbSNPis a collection of SNPs in the human genome. The SNPs in the Factor Hgene are dispersed among the 22 coding exons of the Factor H gene andamong the promoter, the 5′ untranslated region, the introns, and the 3′untranslated region of the Factor H gene. The accession numbers for 379SNPs in the human Factor H gene that are found in the dbSNP database arelisted above. These SNPs can be used in carrying out methods of theinvention.

Five additional polymorphisms in TABLES 11 and 12 are not found in theSNP database: a polymorphism in intron 2 in which two T nucleotides areinserted (IVS2-18insTT); a polymorphism in intron 7 (IVS7-53G>T); apolymorphism in intron 15 (IVS15-30C>A); a polymorphism in intron 18(IVS18-89T>C; and a polymorphism in Exon 20 (N1050Y). Thesepolymorphisms are useful in the methods of the invention. Moreover, itwill be appreciated that these CFHR5 polymorphisms are useful forlinkage and association studies, genotyping clinical populations,correlation of genotype information to phenotype information, loss ofheterozygosity analysis, and identification of the source of a cellsample.

The first row of TABLE 11 lists the exon or intron position of the SNPin the Factor H gene. For exon SNPs, the amino acid position and change,if any, is listed. For example the exon 2 SNP is at position 62 of theFactor H polypeptide and a change from valine (V) to isoleucine (I). Forintron SNPs, the nature the SNP is indicated. For example, the intron 2SNP is an insertion of two nucleotides, TT. The second row of TABLE 11lists dbSNP number, if any, for the polymorphism. For example, rs800292is the dbSNP designation for a polymorphism in exon 2 in the Factor Hgene. A description of this polymorphism, as well as the other FactorH(CFH) gene polymorphisms in dbSNP, is available athttp://www.ncbi.nlm.nih.gov(http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=snp&cmd=search&term=).The third to fifth rows of TABLE 11 lists number of times a particulardiplotype is present among the 22 MPGNII patients. For example, for theexon 2 SNP, GG is present in 20 patients, GA is present in 2 patient,and AA is present in no patients, with MPGNII. The sixth and seventhrows of TABLE 11 list the frequency that a particular haplotype ispresent among the 22 MPGNII patients. For example, for the exon 2 SNP, Gis present in 95%, and A is present in 5%, of the alleles of the 22MPGNII patients. The eighth row lists the nucleotide of the commonhaplotype in the Factor H gene for the 22 MPGNII patients. For example,G is the more frequent nucleotide in the exon 2 SNP, and 9 T nucleotidesis more frequently observed than 11 T nucleotides in the intron 2 SNP,in the Factor H gene for the 22 MPGNII patients. The remaining rows listthe diplotype for the 11 SNPs in the Factor H gene for each of the 22MPGNII patients.

It should be understood that additional polymorphic sites in the FactorH gene, which are not listed in TABLE 11 may be associated with MPGNII.Exemplary polymorphic sites in the Factor H gene are listed, for exampleand not limitation, above.

TABLE 12 shows a comparison of the SNP frequencies in patients withMPGNII versus AMD-negative, ethnically-matched control individuals. Thefirst column of TABLE 12 lists the SNP in the Factor H gene. The secondand third columns of TABLE 12 list the frequencies that a particularhaplotype is present among the 22 MPGNII patients. The fourth and fifthcolumns of TABLE 12 list the frequencies that a particular haplotype ispresent among the 131 control individuals. The sixth column of TABLE 12lists the P-value calculated for each data set.

As shown in TABLES 11 and 12, two frequent non-synonymous variants, 162Vin exon 2 and Y420H in exon 9, a synonymous variant, A307A in exon 10,and a polymorphism in intron 2 exhibited significant association withMPGNII.

As shown in TABLES 14 and 15, a significant association of polymorphicsites in the CHFR5 gene with MPGNII was found in an examination of 22MPGNII cases and 103 ethnically-matched controls. Five (5) polymorphismsin the CFHR5 gene are listed in TABLES 14 and 15; these are found indbSNP in the NCBI. The SNPs in the CFHR5 gene are dispersed among the 10coding exons of the CFHR5 gene and among the promoter, the 5′untranslated region, the introns, and the 3′ untranslated region of theCFHR5 gene. Listed below are the accession numbers for 82 SNPs in thehuman CFHR5 gene that are found in the dbSNP database. These SNPs can beused in carrying out methods of the invention.

TABLE B rs16840956 rs12116643 rs10922151 rs9427662 rs7535993 rs6694672rs1332664 rs16840946 rs12097879rs rs10801584 rs9427661 rs7532441rs6692162 rs1325926 rs16840943 12097550 rs10801583 rs9427660 rs7532068rs6657256 rs1170883 rs12755054 rs12092294 rs10622350 rs9427659 rs7528757rs6657171 rs1170882 rs12750576 rs12091602 rs10614978 rs7555407 rs7527910rs5779855 rs1170881 rs12745733 rs12064805 rs10613146 rs7555391 rs7522952rs3748557 rs1170880 rs12736097 rs12049041 rs10588279 rs7554757 rs7522197rs2151137 rs1170879 rs12736087 rs12039272 rs9727516 rs7550970 rs7419075rs2151136 rs1170878 rs12735776 rs11583363 rs9427942 rs7550735 rs7366339rs1855116 rs928440 rs12731848 rs11306823 rs9427941 rs7550650 rs6702632rs1759016 rs928439 rs12731209 rs10922153 rs9427664 rs7547265 rs6702340rs1750311 rs12142971 rs10922152 rs9427663 rs7537588 rs6674853 rs1412636

The first row of TABLE 14 lists the exon, promoter or intron position ofthe SNP in the CFHR5 gene. For exon SNPs, the amino acid position andchange, if any, is listed. For example, the exon 2 SNP is at position 46of the CFHR5 polypeptide and a change from proline (P) to serine (S).For promoter and intron SNPs, the nature of the SNP is indicated. Forexample, the promoter SNP at position −249 replaces T with C. The secondrow of TABLE 14 lists dbSNP number, if any, for the polymorphism. Forexample, rs9427661 is the dbSNP designation for a polymorphism in thepromoter region of the CFHR5 gene. A description of this polymorphism,as well as the other CFHR5 gene polymorphisms in dbSNP, is available athttp://www.ncbi.nlm.nih.gov(http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=snp&cmd=search&term=).The third to fifth rows of TABLE 14 lists number of times a particulardiplotype is present among the 22 MPGNII patients. For example, for theexon 2 SNP, CC is present in 19 patients, CT is present in 3 patients,and TT is present in no patients, with MPGNII. The sixth and seventhrows of TABLE 14 list the frequency that a particular haplotype ispresent among the 22 MPGNII patients. For example, for the exon 2 SNP, C(encoding proline) is present in 93%, and T (encoding serine) is presentin 7%, of the alleles of the 22 MPGNII patients. The eighth row liststhe nucleotide of the common haplotype in the CFHR5 gene for the 22MPGNII patients. For example, C is the more frequent nucleotide in theexon 2 SNP in the CFHR5 gene for the 22 MPGNII patients. The remainingrows list the diplotype for the 5 SNPs in the CFHR5 gene for each of the22 MPGNII patients.

It should be understood that additional polymorphic sites in the CFHR5gene, which are not listed in TABLE 14 may be associated with MPGNII.Exemplary polymorphic sites in the CFHR5 gene are listed, for exampleand not limitation, above.

TABLE 15 shows a comparison of the SNP frequencies in patients withMPGNII versus AMD-negative, ethnically-matched control individuals. Thefirst column of TABLE 15 lists the SNP in the CFHR5 gene. The second andthird columns of TABLE 15 list the frequencies that a particularhaplotype is present among the 22 MPGNII patients. The fourth and fifthcolumns of TABLE 15 list the frequencies that a particular haplotype ispresent among the 103 control individuals. The sixth column of TABLE 15lists the P-value calculated for each data set.

As shown in TABLES 14 and 15, one non-synonymous variant, P46S in exon2, and two promoter polymorphisms, −249T>C and −2-T>C, exhibitedsignificant association with MPGNII.

Risk-Associated (“Risk”) Polymorphisms and Haplotypes Identified InMPGNII Patients

Sites comprising polymorphisms in Factor H and CFHR5 associated withincreased risk for MPGNII are shown in TABLES 11 and 12 and TABLES 14and 15, respectively. Polymorphisms particularly associated withincreased risk in Factor H and CFHR5 include a variant allele atrs1061170 (Y420H in exon 9) and rs12097550 (P46S in exon 2),respectively.

Certain haplotypes associated with increased risk for MPGNII are shownin TABLES 12 and 15. As shown in TABLE 12, one at-risk haplotype in theFactor H gene includes the variant allele (encoding histidine) atposition 402 and is found in 64% of MPGNII cases, but only in 33% ofcontrols. As shown in TABLE 15, one at-risk haplotype in the CFHR5 geneincludes the variant allele (encoding serine) at position 46 and isfound in 7% of MPGNII cases, but only in <1% of controls.

It will be appreciated that additional polymorphic sites in the Factor Hand CFHR5 genes, which are not listed in TABLES 11-12 and 14-15, mayfurther refine these haplotype analyses. A haplotype analysis usingnon-synonymous polymorphisms in the Factor H or CFHR5 gene is useful toidentify variant Factor H or CFHR5 polypeptides. Other haplotypesassociated with risk may encode a protein with the same sequence as aprotein encoded by a neutral or protective haplotype, but contain anallele in a promoter or intron, for example, that changes the level orsite of Factor H or CFHR5 expression.

Protective Polymorphisms and Haplotypes

Unexpectedly, protective polymorphisms and haplotypes were alsodiscovered. For example, as shown in TABLE 12, the haplotype with thevariant allele in exon 2 (rs800292, 162V) occurs in 23% of controls, butonly in <3% of MPGNII cases and the haplotype with the variant allele inIVS2 (intron 2, −18 insTT) occurs in 26% of controls, but only in <3% ofMPGNII cases. The haplotype with the variant allele in exon 10(rs2274700, A473A) occurs at higher frequency in controls than in MPGNIIcases.

In some cases the protein encoded by a gene characterized by aprotective haplotype has a sequence different from risk haplotypeproteins. For example, a protective form of Factor H protein generallydoes not have histidine at position 402. In some embodiments aprotective form has isoleucine at position 62. Additional protectiveforms can be identified by (1) identifying an individual or individualswith a protective haplotype and (2) determining the sequence(s) ofFactor H cDNA or protein from the individuals. Some protective forms areless than full-length. Protective forms of CFHR5 protein may besimilarly identified.

Neutral Polymorphisms and Haplotypes

Certain haplotypes are associated with neither increased risk ordecreased risk of developing MPGNII and are referred to as “neutral.”Proteins encoded by a gene characterized by a neutral haplotype are“neutral” Factor H or CFHR5 proteins. For example, exemplary proteinsencoded by genes characterized by a neutral haplotype include Factor Hproteins not having histidine at position 402 or isoleucine at position62, and CFHR5 proteins not having serine at position 46.

Significance of Polymorphisms in MPGNII Patients

As shown in Example 2, it has been discovered that the same CFHpolymorphisms associated with propensity to develop AMD are alsoassociated with development of membranoproliferative glomerulonephritistype 2 (MPGN II). Indeed, the risk haplotypes originally found in AMDpatients (Y402H and IVS10) are also found in 70% of patients testedhaving membranoproliferative glomerulonephritis type 2 (MPGN II),indicating that the diagnostic methods of the invention are useful todetect this condition. In addition, variations and haplotypes in theCFHR5 gene were strongly associated with increased risk of havingMPGNII. One conclusion that emerges from these data is that MPGNII andAMD are alternative manifestations of the same genetic lesion. Notably,patients with MPGNII develop drusen that are clinically andcompositionally indistinguishable from drusen that form in AMD. Thesingle feature that distinguishes these two fundus phenotypes is age ofonset—drusen in MPGNII develop early, often in the second decade oflife, while drusen in AMD develop later in life. We conclude thatpolymorphisms in the Factor H gene and CFHR5 gene identified in eitherpopulation (AMD or MPGNII) are predictive of susceptibility to bothdiseases. There are likely other factors that contribute to MPGNII andaccount for the early manifestation. Because AMD is very common andMPGNII is rare, the haplotype analysis of both CFH and CFHR5 genes andother methods described herein will be useful for screening andtreatment of patients with AMD, or with an increased likelihood ofdeveloping AMD.

Loss of Function

Loss of the normal or wild-type function of Factor H or CFHR5 may beassociated with AMD. Non-synonymous polymorphisms in the Factor H gene,such as those shown in TABLES 1A, 1B, 1C, 11, 14 and 15, showing thestrongest correlation with AMD and resulting in a variant Factor Hpolypeptide or variant CFHR5 polypeptide, are likely to have a causativerole in AMD. Such a role can be confirmed by producing a transgenicnon-human animal expressing human Factor H or CFHR5 bearing such anon-synonymous polymorphism(s) and determining whether the animaldevelops AMD. Polymorphisms in Factor H or CFHR5 coding regions thatintroduce stop codons may cause AMD by reducing or eliminatingfunctional Factor H or CFHR5 protein. Stop codons may also causeproduction of a truncated Factor H or CFHR5 peptide with aberrantactivities relative to the full-length protein. Polymorphisms inregulatory regions, such as promoters and introns, may cause AMD bydecreasing Factor H or CFHR5 gene expression. Polymorphisms in introns(e.g., intron 2 of CFH) may also cause AMD by altering gene splicingpatterns resulting in an altered Factor H or CFHR5 protein. CFH RNA orproteins can be assayed to detect changes in expression of splicevariants, where said changes are indicative of a propensity to developAMD. Alternative splice patterns have been reported for the Factor Hgene itself.

The effect of polymorphisms in the Factor H gene or CFHR5 gene on AMDcan be determined by several means. Alterations in expression levels ofa variant Factor H or CFHR5 polypeptide can be determined by measuringprotein levels in samples from groups of individuals having or nothaving AMD or various subtypes of AMD. Alterations in biologicalactivity of variant Factor H or CFHR5 polypeptides can be detected byassaying for in vitro activities of Factor H or CFHR5, for example,binding to C3b or to heparin, in samples from the above groups ofindividuals.

VI. Polymorphisms at Sites of Genomic Duplication

As illustrated in FIG. 18, the genes for CFH and the factor H related(CFHR) 1-5 genes have regions of shared, highly conserved, sequencewhich likely arose from genomic duplications. Certain SNPs andvariations found in CFH or CFHR5, such as those described herein, arealso expected in the corresponding sequences of CFHR1, CFHR2, CFHR3, andCFHR4. For example, sequences corresponding to CFH exon 22 are found inCFH, CFHR1 and CFHR2, and it is possible that polymorphisms identifiedin exon 22 of CFH (e.g., R1210C) are also found in CFHR1 and/or CFHR2and these variants might be linked to propensity to development of AMD,MPGNII, and other complement related conditions. Homologous blocks ofsequence flanking the polymorphic sites identified in CFH and CFHR5 canbe identified by alignment of the cDNA or genomic sequences in thoseregions. The conserved sequences flanking the polymorphic site usuallycomprise at least 10 bp (on either side of the polymorphic site) andmore often at least 20 bp, or at least 50 bp, or at least 100 bp with atleast 95% identity at the nucleotide level, and sometimes with at least98% identity, at least 99% identity, or even 100% identity). Identitycan be determined by inspection or using well know algorithms (Smith andWaterman, 1981 or Needleman and Wunsch, 1970, both supra). The inventiontherefore provides methods of determining a subject's propensity todevelop age-related macular degeneration (AMD) or other conditions bydetecting the presence or absence of a variation at a polymorphic siteof a Factor H-related gene that corresponds to a homologous polymorphicsite in the CFH or CFHR5 gene.

Sequences for CFH and the factor H related genes are known in the art(see sequences and accession numbers provided elsewhere herein). Alsosee Rodriquez de Cordoba, S., et al, 2004, Mol Immunol 41:355-67; Zipfelet al, 1999, Immunopharmocology 42:53-60; Zipfel et al., Factor H familyproteins: on complement, microbes and human diseases, Biochem Soc Trans.2002 November; 30(Pt 6):971-8; Diaz-Guillen M A, et al., A radiationhybrid map of complement factor H and factor H-related genes,Immunogenetics, 1999 June; 49(6):549-52; Skerka C, et al., A novel shortconsensus repeat-containing molecule is related to human complementfactor H, J Biol Chem. 1993 Feb. 5; 268(4):2904-8; Skerka C, et al., Thehuman factor H-related gene 2 (FHR2): structure and linkage to thecoagulation factor XIIIb gene, Immunogenetics, 1995; 42(4):268-74; MaleD A, et al., Complement factor H: sequence analysis of 221 kb of humangenomic DNA containing the entire fH, fHR-1 and fHR-3 genes, MolImmunol. 2000 January-February; 37(1-2):41-52; Hellwage J, et al.,Biochemical and functional characterization of the factor-H-relatedprotein 4 (FHR-4), Immunopharmacology. 1997 December; 38(1-2):149-57;Skerka C, et al., The human factor H-related protein 4 (FHR-4). A novelshort consensus repeat-containing protein is associated with humantriglyceride-rich lipoproteins, J Biol Chem. 1997 Feb. 28;272(9):5627-34; Hellwage J, et al., Functional properties of complementfactor H-related proteins FHR-3 and FHR-4: binding to the C3d region ofC3b and differential regulation by heparin, FEBS Lett. 1999 Dec. 3;462(3):345-52; Jozsi M, et al., FHR-4A: a new factor H-related proteinis encoded by the human FHR-4 gene, Eur J Hum Genet. 2005 March;13(3):321-9; McRae J L, et al., Location and structure of the humanFHR-5 gene, Genetica. 2002 March; 114(2):157-61; McRae J L, et al.,Human factor H-related protein 5 has cofactor activity, inhibits C3convertase activity, binds heparin and C-reactive protein, andassociates with lipoprotein, J Immunol. 2005 May 15; 174(10):6250-6;Murphy B, et al., Factor H-related protein-5: a novel component of humanglomerular immune deposits, Am J Kidney Dis. 2002 January; 39(1):24-7.

VII. Detection and Analysis of Factor H Polymorphisms Associated withAMD

The discovery that polymorphic sites and haplotypes in the Factor H geneand CFHR5 gene are associated with AMD (and MPGNII) has a number ofspecific applications, including screening individuals to ascertain riskof developing AMD and identification of new and optimal therapeuticapproaches for individuals afflicted with, or at increased risk ofdeveloping, AMD. Without intending to be limited to a specificmechanism, polymorphisms in the Factor H gene may contribute to thephenotype of an individual in different ways. Polymorphisms that occurwithin the protein coding region of Factor H may contribute to phenotypeby affecting the protein structure and/or function. Polymorphisms thatoccur in the non-coding regions of Factor H may exert phenotypic effectsindirectly via their influence on replication, transcription and/ortranslation. Certain polymorphisms in the Factor H gene may predisposean individual to a distinct mutation that is causally related to aparticular AMD phenotype. Alternatively, as noted above, a polymorphismin the CFH gene, or in a CFHR5, may be linked to a variation in aneighboring gene (including but not limited to CFHR-1, 2, 3, or 4). Thevariation in the neighboring gene may result in a change in expressionor form of an encoded protein and have detrimental or protective effectsin the carrier.

A. Preparation of Samples for Analysis

Polymorphisms are detected in a target nucleic acid isolated from anindividual being assessed. Typically genomic DNA is analyzed. For assayof genomic DNA, virtually any biological sample containing genomic DNAor RNA, e.g., nucleated cells, is suitable. For example, in theexperiments described in Example 1, genomic DNA was obtained fromperipheral blood leukocytes collected from case and control subjects(QIAamp DNA Blood Maxi kit, Qiagen, Valencia, Calif.). Other suitablesamples include saliva, cheek scrapings, biopsies of retina, kidney orliver or other organs or tissues; skin biopsies; amniotic fluid or CVSsamples; and the like. Alternatively RNA or cDNA can be assayed.Alternatively, as discussed below, the assay can detect variant Factor Hproteins. Methods for purification or partial purification of nucleicacids or proteins from patient samples for use in diagnostic or otherassays are well known.

B. Detection of Polymorphisms in Target Nucleic Acids

The identity of bases occupying the polymorphic sites in the Factor Hgene and the Factor H-Related 5 gene shown in TABLES 1A, 1B, 1C, 11, 14and 15, as well as others in the dbSNP collection that are located in oradjacent to the Factor H or CFHR5 genes (see lists above), can bedetermined in an individual, e.g., in a patient being analyzed, usingany of several methods known in the art. Examples include: use ofallele-specific probes; use of allele-specific primers; direct sequenceanalysis; denaturing gradient gel electrophoresis (DGGE) analysis;single-strand conformation polymorphism (SSCP) analysis; and denaturinghigh performance liquid chromatography (DHPLC) analysis. Other wellknown methods to detect polymorphisms in DNA include use of: MolecularBeacons technology (see, e.g., Piatek et al., 1998; Nat. Biotechnol.16:359-63; Tyagi, and Kramer, 1996, Nat. Biotechnology 14:303-308; andTyagi, et al., 1998, Nat. Biotechnol. 16:49-53), Invader technology(see, e.g., Neri et al., 2000, Advances in Nucleic Acid and ProteinAnalysis 3826:117-125 and U.S. Pat. No. 6,706,471), nucleic acidsequence based amplification (Nasba) (Compton, 1991), Scorpiontechnology (Thelwell et al., 2000, Nuc. Acids Res, 28:3752-3761 andSolinas et al., 2001, “Duplex Scorpion primers in SNP analysis and FRETapplications” Nuc. Acids Res, 29:20.), restriction fragment lengthpolymorphism (RFLP) analysis, and the like. Additional methods will beapparent to the one of skill.

The design and use of allele-specific probes for analyzing polymorphismsare described by e.g., Saiki et al., 1986; Dattagupta, EP 235,726,Saiki, WO 89/11548. Briefly, allele-specific probes are designed tohybridize to a segment of target DNA from one individual but not to thecorresponding segment from another individual, if the two segmentsrepresent different polymorphic forms. Hybridization conditions arechosen that are sufficiently stringent so that a given probe essentiallyhybridizes to only one of two alleles. Typically, allele-specific probesare designed to hybridize to a segment of target DNA such that thepolymorphic site aligns with a central position of the probe.

Exemplary allele-specific probes for analyzing Factor H polymorphismsare shown in TABLE 16A. Using the polymorphism dbSNP No. rs1061170 as anillustration, examples of allele-specific probes include:5′-TTTCTTCCATAATTTTG-3′ [SEQ ID NO:234] (reference allele probe) and5′-TTTCTTCCATGATTTTG-3′ [SEQ ID NO:235] (variant allele probe); and5′-TAATCAAAATTATGGAA-3′ [SEQ ID NO:232] (reference allele probe) and5′-TAATCAAAATCATGGAA-3′ [SEQ ID NO:233] (variant allele probe). In thisexample, the first set of allele-specific probes hybridize to thenon-coding strand of the Factor H gene spanning the exon 9 polymorphism.The second set of allele-specific probes hybridize to the coding strandof the Factor H spanning the exon 9 polymorphism. These probes are 17bases in length. The optimum lengths of allele-specific probes can bereadily determined using methods known in the art.

Allele-specific probes are often used in pairs, one member of a pairdesigned to hybridize to the reference allele of a target sequence andthe other member designed to hybridize to the variant allele. Severalpairs of probes can be immobilized on the same support for simultaneousanalysis of multiple polymorphisms within the same target gene sequence.

The design and use of allele-specific primers for analyzingpolymorphisms are described by, e.g., WO 93/22456 and Gibbs, 1989.Briefly, allele-specific primers are designed to hybridize to a site ontarget DNA overlapping a polymorphism and to prime DNA amplificationaccording to standard PCR protocols only when the primer exhibitsperfect complementarity to the particular allelic form. A single-basemismatch prevents DNA amplification and no detectable PCR product isformed. The method works best when the polymorphic site is at theextreme 3′-end of the primer, because this position is mostdestabilizing to elongation from the primer.

Exemplary allele-specific primers for analyzing Factor H polymorphismsare shown in TABLE 16B. Using the polymorphism dbSNP No. rs1061170 as anillustration, examples of allele-specific primers include:5′-CAAACTTTCTTCCATA-3′ [SEQ ID NO:294] (reference allele primer) and5′-CAAACTTTCTTCCATG-3′ [SEQ ID NO:295] (variant allele primer); and5′-GGATATAATCAAAATT-3′ [SEQ ID NO:292] (reference allele primer) and5′-GGATATAATCAAAATC-3′ [SEQ ID NO:293] (variant allele primer). In thisexample, the first set of allele-specific primers hybridize to thenon-coding strand of the Factor H gene directly adjacent to thepolymorphism in exon 9, with the last nucleotide complementary to thereference or variant polymorphic allele as indicated. These primers areused in standard PCR protocols in conjunction with another common primerthat hybridizes to the coding strand of the Factor H gene at a specifiedlocation downstream from the polymorphism. The second set ofallele-specific primers hybridize to the coding strand of the Factor Hgene directly adjacent to the polymorphic site in exon 9, with the lastnucleotide complementary to the reference or variant polymorphic alleleas indicated. These primers are used in standard PCR protocols inconjunction with another common primer that hybridizes to the non-codingstrand of the Factor H gene at a specified location upstream from thepolymorphism. The common primers are chosen such that the resulting PCRproducts can vary from about 100 to about 300 bases in length, or about150 to about 250 bases in length, although smaller (about 50 to about100 bases in length) or larger (about 300 to about 500 bases in length)PCR products are possible. The length of the primers can vary from about10 to 30 bases in length, or about 15 to 25 bases in length. Thesequences of the common primers can be determined by inspection of theFactor H genomic sequence, which is found under GenBank accession numberAL049744.

Many of the methods for detecting polymorphisms involve amplifying DNAor RNA from target samples (e.g., amplifying the segments of the FactorH gene of an individual using Factor H-specific primers) and analyzingthe amplified gene. This can be accomplished by standard polymerasechain reaction (PCR & RT-PCR) protocols or other methods known in theart. The amplifying may result in the generation of Factor Hallele-specific oligonucleotides, which span the single nucleotidepolymorphic sites in the Factor H gene. The Factor H-specific primersequences and Factor H allele-specific oligonucleotides may be derivedfrom the coding (exons) or non-coding (promoter, 5′ untranslated,introns or 3′ untranslated) regions of the Factor H gene.

Amplification products generated using PCR can be analyzed by the use ofdenaturing gradient gel electrophoresis (DGGE). Different alleles can beidentified based on sequence-dependent melting properties andelectrophoretic migration in solution. See Erlich, ed., PCR Technology,Principles and Applications for DNA Amplification, Chapter 7 (W.H.Freeman and Co, New York, 1992).

Alleles of target sequences can be differentiated using single-strandconformation polymorphism (SSCP) analysis. Different alleles can beidentified based on sequence- and structure-dependent electrophoreticmigration of single stranded PCR products (Orita et al., 1989).Amplified PCR products can be generated according to standard protocols,and heated or otherwise denatured to form single stranded products,which may refold or form secondary structures that are partiallydependent on base sequence.

Alleles of target sequences can be differentiated using denaturing highperformance liquid chromatography (DHPLC) analysis. Different allelescan be identified based on base differences by alteration inchromatographic migration of single stranded PCR products (Frueh andNoyer-Weidner, 2003). Amplified PCR products can be generated accordingto standard protocols, and heated or otherwise denatured to form singlestranded products, which may refold or form secondary structures thatare partially dependent on the base sequence.

Direct sequence analysis of polymorphisms can be accomplished using DNAsequencing procedures that are well-known in the art. See Sambrook etal., Molecular Cloning, A Laboratory Manual (2nd Ed., CSHP, New York1989) and Zyskind et al., Recombinant DNA Laboratory Manual (Acad.Press, 1988).

A wide variety of other methods are known in the art for detectingpolymorphisms in a biological sample. See, e.g., Ullman et al. “Methodsfor single nucleotide polymorphism detection” U.S. Pat. No. 6,632,606;Shi, 2002, “Technologies for individual genotyping: detection of geneticpolymorphisms in drug targets and disease genes” Am J Pharmacogenomics2:197-205; Kwok et al., 2003, “Detection of single nucleotidepolymorphisms” Curr Issues Biol. 5:43-60).

It will be apparent to the skilled practitioner guided by thisdisclosure than various polymorphisms and haplotypes can be detected toassess the propensity of an individual to develop a Factor H relatedcondition. The following examples and combinations, and others providedherein, are provided for illustration and not limitation. In one aspectof the invention, the allele of the patient at one of more of thefollowing polymorphic sites in the Factor H gene is determined:rs529825; rs800292; rs3766404; rs1061147; rs1061170; and rs203674. Inone embodiment the allele of the patient at rs529825 is determined. Inone embodiment the allele of the patient at rs800292 is determined. Inone embodiment the allele of the patient at rs3766404 is determined. Inone embodiment the allele of the patient at rs1061147 is determined. Inone embodiment the allele of the patient at rs1061170 is determined. Inone embodiment the allele of the patient at rs203674 is determined. Inone embodiment at least one of rs529825 and rs800292 is determined. Inone embodiment at least one of rs1061147, rs1061170 and rs203674 isdetermined. In one embodiment at least one of rs529825 and rs800292 isdetermined, rs3766404 is determined, and at least one of rs1061147,rs1061170 and rs203674 is determined. In one embodiment alleles atrs529825, rs800292, rs3766404, rs1061170 and rs203674 are determined.The aforementioned polymorphisms and combinations of polymorphisms areprovided herein for illustration and are not intended to limit theinvention in any way. That is, other polymorphisms and haplotypes usefulin practicing the invention will be apparent from this disclosure.

In a related aspect of the invention, the allele of the patient at oneof more of the following polymorphic sites in the Factor H gene isdetermined: rs529825; rs800292; intron 2 (IVS2 or insTT); rs3766404;rs1061147; rs1061170; exon 10A; rs203674; rs375046; and exon 22 (1210).In one embodiment the allele of the patient at rs529825 is determined.In one embodiment the allele of the patient at rs800292 is determined.In one embodiment the allele of the patient at intron 2 is determined.In one embodiment the allele of the patient at rs3766404 is determined.In one embodiment the allele of the patient at rs1061147 is determined.In one embodiment the allele of the patient at rs1061170 is determined.In one embodiment the allele of the patient at exon 10A is determined.In one embodiment the allele of the patient at rs203674 is determined.In one embodiment the allele of the patient at rs375046 is determined.In one embodiment the allele of the patient at exon 22 (1210) isdetermined. In one embodiment at least one of rs529825 and rs800292 isdetermined; intron 2 is determined; rs3766404 is determined; at leastone of rs10611147, rs10611170 and rs203674 is determined; exon 10A isdetermined; rs375046 is determined; and exon 22 (1210) is determined. Inone embodiment alleles at rs529825, rs800292, intron 2; rs3766404,rs1061170, exon 10A, rs203674, rs375046, and exon 22 (1210) aredetermined. In one embodiment one, two, three, four five, or more thanfive of the following polymorphic sites in the Factor H gene isdetermined: rs529825; rs800292; intron 2 (IVS2 or insTT); rs3766404;rs1061147; rs1061170; rs2274700; exon 10A; rs203674; rs375046; and exon22 (1210). The aforementioned polymorphisms and combinations ofpolymorphisms are provided for illustration and are not intended tolimit the invention in any way.

As discussed above, the non-synonymous polymorphism at amino acidposition 1210 in exon 22 of the Factor H gene is strongly associatedwith AMD, and the presence of cysteine at amino acid position 1210 ofFactor H, therefore, provides a strong indication that the individualhas AMD or is likely to develop AMD. Remarkably, 1210C is indicative ofpropensity to develop AMD or other complement mediated conditions evenwhen detected on allele that is otherwise protective (e.g., Y402). Thus,the allele of the patient at exon 22 (1210) is highly informative withrespect to risk of developing AMD or other Factor H-associated diseases.

In a related aspect of the invention, the allele of an individual at oneof more of the following polymorphic sites in the CFHR5 gene isdetermined: rs9427661 (−249T>C); rs9427662 (−20T>C); and rs12097550(P46S). In one embodiment the allele of the patient at rs9427661 isdetermined. In one embodiment the allele of the patient at rs9427662 isdetermined. In one embodiment the allele of the patient at rs12097550 isdetermined. In one embodiment at least one of rs9427661 and rs9427662 isdetermined. In one embodiment at least one of rs9427661 and rs9427662 isdetermined, and rs12097550 is determined. In one embodiment rs9427661,rs9427662 and rs12097550 is determined. The aforementioned polymorphismsand combinations of polymorphisms are provided for illustration and arenot intended to limit the invention in any way. That is, otherpolymorphisms and haplotypes useful in practicing the invention will beapparent from this disclosure.

C. Detection of Protein Variants

In one embodiment of the invention, a protein assay is carried out tocharacterize polymorphisms in a subject's CFH or CFHR5 genes. Methodsthat can be adapted for detection of variant CFH, HFL1 and CFHR5 arewell known. These methods include analytical biochemical methods such aselectrophoresis (including capillary electrophoresis and two-dimensionalelectrophoresis), chromatographic methods such as high performanceliquid chromatography (HPLC), thin layer chromatography (TLC),hyperdiffusion chromatography, mass spectrometry, and variousimmunological methods such as fluid or gel precipitin reactions,immunodiffusion (single or double), immunoelectrophoresis,radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs),immunofluorescent assays, western blotting and others.

For example, a number of well established immunological binding assayformats suitable for the practice of the invention are known (see, e.g.,Harlow, E.; Lane, D. Antibodies: a laboratory manual. Cold SpringHarbor, N.Y.: Cold Spring Harbor Laboratory; 1988; and Ausubel et al.,(2004) Current Protocols in Molecular Biology, John Wiley & Sons, NewYork N.Y. The assay may be, for example, competitive or non-competitive.Typically, immunological binding assays (or immunoassays) utilize a“capture agent” to specifically bind to and, often, immobilize theanalyte. In one embodiment, the capture agent is a moiety thatspecifically binds to a variant CFH or CFHR5 polypeptide or subsequence.The bound protein may be detected using, for example, a detectablylabeled anti-CFH/CFHR5 antibody. In one embodiment, at least one of theantibodies is specific for the variant form (e.g., does not bind to thewild-type CFH or CFHR5 polypeptide. In one embodiment, the variantpolypeptide is detected using an immunoblot (Western blot) format.

D. Patient Screening/Diagnosis of AMD

Polymorphisms in the Factor H gene, such as those shown in TABLE 1A,TABLE 1B, TABLE 1C or identified as described herein, which correlatewith AMD or with particular subtypes of AMD, are useful in diagnosingAMD or specific subtypes of AMD, or susceptibility thereto.Polymorphisms in the CFHR5 gene, such as those shown in TABLES 14 and 15or identified as described herein, which correlate with AMD or withparticular subtypes of AMD, are useful in diagnosing AMD or specificsubtypes of AMD, or susceptibility thereto. These polymorphisms are alsouseful for screening for MPGNII and other Factor H-associated diseases.

Individuals identified as at high risk for developing AMD can take stepsto reduce risk, including frequent opthalmological examinations andtreatments described below, known in the art, or developed in thefuture.

As described in Example 1, an at-risk CFH haplotype in combination witha triggering event (e.g., infection) appears to be sufficient fordisease manifestation. Patients identified as at-risk for AMD canreceive aggressive therapy (e.g., using antibiotics, anti-inflamatoryagents, treatment with protective forms of CFH/CFHR5, or treatment withother modulators of CFH activity) at early signs of infection.

Combined detection of several such polymorphic forms (i.e., the presenceor absence of polymorphisms at specified sited), for example, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or all of the polymorphisms in the Factor H genelisted in TABLE 1A, TABLE 1B and/or TABLE 1C, alone or in combinationwith additional Factor H gene polymorphisms not included in TABLES1A-1C, may increase the probability of an accurate diagnosis. Similarly,combined detection of several polymorphic forms in the CFHR5 gene, forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of the polymorphisms inthe CHFR5 gene listed in TABLES 14 and 15, alone or in combination withadditional CFHR5 gene polymorphisms not included in TABLES 14 and 15,may increase the probability of an accurate diagnosis. In oneembodiment, screening involves determining the presence or absence of atleast one polymorphism in the Factor H gene and at least onepolymorphism in the CFHR5 gene. In one embodiment, screening involvesdetermining the presence or absence of at least 2, 3, or 4 polymorphismsin the Factor H gene in combination with and at least 2, 3, or 4polymorphisms in the CFHR5 gene.

The polymorphisms in the Factor H and CFHR5 genes are useful indiagnosing AMD or specific subtypes of AMD, or susceptibility thereto,in family members of patients with AMD, as well as in the generalpopulation.

In diagnostic methods, analysis of Factor H polymorphisms and/or CFHR5polymorphisms can be combined with analysis of polymorphisms in othergenes associated with AMD, detection of protein markers of AMD (see,e.g., Hageman et al., patent publications US20030017501; US20020102581;WO0184149; and WO0106262), assessment of other risk factors of AMD (suchas family history), with opthalmological examination, and with otherassays and procedures.

E) Identification of Patients for Drug Therapy

Polymorphisms in the Factor H gene and CFHR5 gene are also useful foridentifying suitable patients for conducting clinical trials for drugcandidates for AMD. Such trials are performed on treated or controlpopulations having similar or identical polymorphic profiles at adefined collection of polymorphic sites in the Factor H gene and/orCFHR5 gene, or having similar or identical Factor H haplotypes and/orCFHR5 haplotypes. The use of genetically matched populations eliminatesor reduces variation in treatment outcome due to genetic factors,leading to a more accurate assessment of the efficacy of a potentialdrug.

F) Screening Donor Tissue for Transplantation

Transplantation of organs (e.g., liver) and tissues (e.g., blood,hepatocytes) is increasingly common. It is desirable, in carrying outsuch transplantation, to avoid introducing into the recipient adeleterious form of Factor H or a Factor H-Related Protein and therebyincreasing the recipient's risk of developing AMD. Thus, in one aspectof the invention, a donor tissue is tested to detect the presence orabsence of a variation at a polymorphic site of a Factor H or CFHR5 geneto identify host tissues carrying risk haplotypes or other deleterioussequences. In addition or alternatively, organs and tissues can betested for the expression of forms of Factor H or CFHR proteins, forexample by using immunological assays as described herein. In oneembodiment the transplanted tissue is blood or plasma (i.e., given in ablood transfusion or plasma replacement). Routine screening of donatedblood to avoid administration of a protein associated with risk (e.g.,the 1210C form of CFH) may avoid compromising the recipient.

G) Phenotypic Categories

Susceptibility to specific subtypes of AMD can be identified based onthe association with particular haplotypes. Thus, the screening can beused to determine suitable therapies for groups of patients withdifferent genetic subtypes of AMD.

The methods may be used for the diagnosis of AMD, which may besubdivided into phenotypic categories (for example, early AMD (ARM)geographic atrophy (GA) and exudative AMD (CNV)). The ARM and GAphenotypes may be further subdivided into distinct phenotypes (forexample, RPE changes alone, >10 macular hard drusen, macular softdrusen, BB (cuticular) drusen, pigment epithelial detachment (PED),“Cherokee” atrophy, peninsular geographic atrophy and pattern geographicatrophy). For descriptions of these phenotypes see, e.g., Bird et al.,1995, Surv Ophthalmol 39, 367-74; and Klayer et al., 2001, InvestOphthalmol Vis Sci 42, 2237-41.

H) Other Diseases

Polymorphisms in the Factor H and CFHR5 gene, such as those shown inTABLES 1A, 1B, 1C, 11, 14 and 15, can also be tested for associationwith other diseases, (for example, Alzheimer's disease, multiplesclerosis, lupus, and asthma) and conditions (for example, burninjuries, transplantation, and stroke) which involve dysregulation ofthe alternative complement pathway, that have known but hithertounmapped genetic components. Without being limited to any particularmechanism of action, it is suggested herein that expression of variantFactor H and/or CFHR5 polypeptides is associated with dysregulation ofthe alternative complement pathway. The variant forms of Factor H and/orCHFR5 may have a causal effect on diseases involving a defect in thealternative complement pathway, or the presence of variant forms ofFactor H and/or CFHR5 may indicate that another gene involved in thealternative complement pathway has a causal effect.

Polymorphisms in the Factor H gene may also be useful in mapping andtreating diseases that map to chromosome 1q, in particular at or near1q32 where the Factor H gene is located. This particular locus containsa number of complement pathway-associated genes. One group of thesegenes, referred to as the regulators of complement activation (RCA) genecluster, contains the genes that encode Factor H, five Factor H-relatedgenes and the beta subunit of coagulation factor XIII. A second clusterof complement-associated genes, including C4BPA, C4BPB, C4BPAL2, DAF(CD55) CR1, CR2, CR1L and MCP (CD46) lies immediately adjacent to the1q25-31 locus.

VIII. Prevention and Treatment of AMD

A patient with a Factor H polymorphism can be treated by administeringto a patient an antagonist of the variant Factor H polypeptide and/orvariant CHFR5 polypeptide. An antagonist may include a therapeuticamount of an RNA complementary to the nucleotide sequence of a variantFactor H polypeptide and/or variant CHFR5 polypeptide or an antibodythat specifically interacts with and neutralizes the activity of avariant Factor H polypeptide and/or variant CHFR5 polypeptide.Alternatively, AMD associated with the Factor H polymorphism and/orCFHR5 polymorphism can be treated by administering to a patient a formof Factor H and/or CHFR5 not associated with increased risk, such as thenormal or wild-type Factor H protein and/or normal or wild-type CHFR5polypeptide. In one method of the invention, a protective variant formof Factor H and/or protective variant form of CHFR5 is administered to apatient.

Therapeutic and prophylactic approaches in subjects identified as beingat high risk for AMD include, but are not limited to, (1) increasing theamount or expression of neutral or protective forms of Factor H and/orneutral or protective forms of CHFR5; (2) decreasing the amount orexpression of risk-associated forms of Factor H and/or risk-associatedform of CHFR5; and (3) reducing activation of the complement alternativepathway. Examples of such therapeutic and prophylactic approachesinclude: (1) administration of neutral or protective forms of Factor Hprotein or therapeutically active fragments and/or neutral or protectiveforms of CHFR5 or therapeutically active fragments; (2) other wiseincreasing expression or activity of neutral and protective forms ofFactor H; (3) interfering with expression of variant Factor H and/orvariant CFHR5 proteins encoded by individuals with a risk haplotype by(e.g., by administration of antisense RNA); (4) reducing the amount ofactivity of a detrimental variant form.

Therapeutic agents (e.g., agents that increase or decrease levels ofwild-type or variant Factor H or modulate its activity and/or agentsthat increase or decrease levels of wild-type or variant CFHR5 ormodulate its activity) can be administered systemically (e.g., by i.v.injection or infusion) or locally (e.g., to the vicinity of the ocularRPE for treatment of AMD). Methods for administration of agents to theeye are well known in the medical arts and can be used to administer AMDtherapeutics described herein. Exemplary methods include intraocularinjection (e.g., retrobulbar, subretinal, intravitreal andintrachoridal), iontophoresis, eye drops, and intraocular implantation(e.g., intravitreal, sub-Tenons and sub-conjunctival). For examples,anti-VEGF antibody has been introduced into cynomolgus monkeys byintravitreal injection (see, e.g., Gaudreault et al., 2005, “Preclinicalpharmacokinetics of Ranibizumab (rhuFabV2) after a single intravitrealadministration” Invest Ophthalmol Vis Sci. 46:726-33), and bioactiveVEGF and bFGF have been expressed in the eye via intravitrealimplantation of sustained release pellets (Wong et al., 2001,“Intravitreal VEGF and bFGF produce florid retinal neovascularizationand hemorrhage in the rabbit” Curr Eye Res. 22:140-7). Importantly, ithas been discovered that Factor H is synthesized locally by the retinalpigment epithelium (see Example 1), indicating that local administrationof agents has therapeutic benefit.

A. Administration of Therapeutic Factor H Polypeptides

Administration of neutral or protective forms of Factor H polypeptidesand/or neutral or protective forms of CFHR5 polypeptides to subjects atrisk for developing AMD (and/or with early stage disease) can be used toameliorate the progression of the disease.

In one approach, recombinant Factor H polypeptide is administered to thepatient. In one embodiment, the recombinant Factor H is encoded by aneutral haplotype sequence, which may be full-length (CFH/HF1),truncated (FHL1), or alternatively spliced form, or a biologicallyactive fragment thereof. In another embodiment the recombinant Factor Hhas the sequence of a protective allele, either full-length or truncatedform, or a protective biologically active fragment thereof. Methods forproduction of therapeutic recombinant proteins are well known andinclude methods described hereinbelow. The therapeutic polypeptide canbe administered systemically (e.g., intravenously or by infusion) orlocally (e.g., directly to an organ or tissue, such as the eye or theliver).

Some protective forms of Factor H and the CHFL1 protein are less thanfull-length. For example, fragments of neutral or protective forms ofFactor H may be administered for treatment or prevention of AMD orMPGNII. In a particular embodiment, polypeptides encoded by CFH splicevariants expressed in individuals with a protected phenotype areadministered. These proteins can be identified by screening expressionof CFH-related RNA in individuals homozygous for a protective or neutralhaplotype.

In particular embodiments, the protective protein has a sequencecorresponding to one or more exons of the CFH gene sequence. Forexample, the protective protein may have the sequence of full-length ortruncated CFH protein, except that the amino acid residues encoded by 1,2, 3 or more exons (which may or may not be contiguous) are deleted.

In one embodiment a protective Factor H protein of the invention has anamino acid sequence substantially identical to SEQ ID NO:2, with theproviso that the residue at position 402 is not histidine and theresidue at position 1210 is not cysteine. In one embodiment the residueat position 62 is not valine. Preferably, the residue at position 62 isisoleucine. Preferably the residue at position 62 is isoleucine, theresidue at position 402 is tyrosine and the residue at position 1210 isarginine. Preferably the protective Factor H protein has 95% amino acididentity to SEQ ID NO:2 or a fragment thereof; sometimes at least 95%amino acid identity, sometimes at least 98% amino acid identity, andsometimes at least 99% identity to the reference Factor H polypeptide ofSEQ ID NO:2. The polypeptide sequence of an exemplary protective variantof human Factor H [SEQ ID NO:5] is shown in FIG. 10. This protectivevariant Factor H polypeptide has an isoleucine at amino acid position 62and a tyrosine at amino acid position 402 (indicated in bold). Thepolypeptide sequence of an exemplary protective variant of HFL1, thetruncated form of human Factor H (SEQ ID NO:6) is shown in FIG. 11. Thisprotective variant truncated Factor H polypeptide has a isoleucine atamino acid position 62 and tyrosine at amino acid position 402(indicated in bold).

In one embodiment a protective Factor H protein of the invention has anamino acid sequence substantially identical to SEQ ID NO:4 (FHL1). Inone embodiment the residue at position 62 is not valine. Preferably, theresidue at position 62 is isoleucine. Preferably the protective Factor Hprotein has 95% amino acid identity to SEQ ID NO:4 or a fragmentthereof; sometimes at least 95% amino acid identity, sometimes at least98% amino acid identity, and sometimes at least 99% identity to thereference Factor H polypeptide of SEQ ID NO:4.

In some embodiments the protective Factor H protein has one or moreactivities of the reference Factor H polypeptide. In one embodiment theactivity is binding to heparin. In one embodiment the activity isbinding to CRP. In one embodiment the activity is binding to C3b. In oneembodiment the activity is binding to endothelial cell surfaces. In oneembodiment the activity is C3b co-factor activity. In one embodiment,the protective Factor H protein has activity that is higher with respectto its normal function than the protein of SEQ ID NO:2. In oneembodiment, the protective Factor H protein has activity with respect toits normal function that is higher than the protein of SEQ ID NO:4.

Assays for Factor H activities are well known and described in thescientific literature. For illustration and not limitation, examples ofassays will be described briefly.

Binding of Protective Proteins (CFH Variants) to C3b or CRP.

Interactions between C3b and CFH proteins can be analyzed by surfaceresonance using a Biacore 3000 system (Biacore AB, Uppsala, Sweden), asdescribed previously (Manuelian et al., 2003, Mutations in factor Hreduce binding affinity to C3b and heparin and surface attachment toendothelial cells in hemolytic uremic syndrome. J Clin Invest 111,1181-90). In brief, C3b (CalBiochem, Inc), are coupled using standardamine-coupling to flow cells of a sensor chip (Carboxylated Dextran ChipCM5, Biacore AB, Uppsala, Sweden). Two cells are activated and C3b (50μg/ml, dialyzed against 10 mM acetate buffer, pH 5.0) is injected intoone flow cell until a level of coupling corresponding to 4000 resonanceunits is reached. Unreacted groups are inactivated usingethanolamine-HCl. The other cell is prepared as a reference cell byinjecting the coupling buffer without C3b. Before each binding assay,flow cells will be washed thoroughly by two injections of 2 M NaCl in 10mM acetate buffer, pH 4.6 and running buffer (PBS, pH 7.4). The Factor Hprotein is injected into the flow cell coupled with C3b or into thecontrol cell at a flow rate of 5 ul/min at 25° C. Binding of Factor H toC3b is quantified by measuring resonance units over time, as describedin Manuelian et al., 2003, supra.

Interactions between CRP and CHF proteins can be analyzed by surfaceresonance in an identical manner by substituting CRP for C3b in flowcells of a sensor chip

Binding to Endothelial Cell Surface

Binding of CHF proteins to endothelial cell surfaces is assayed byimmunofluorescence staining of HUVECs and FACS analysis. HUVEC cells arekept in serum free DMEM (BioWhittaker) for 24 hrs prior to the assay.Cells are detached from the surface with DPBS/EDTA and washed twice withDPBS; 5×10⁵ cells will be transferred into plastic tubes and unspecificbinding sites will be blocked with 1% BSA/DPBS for 15 min prior toincubation with purified allele variants of factor H (5 μg). Controlsare performed in the absence of the factor H isoform. Following bindingof factor H, cells are thoroughly washed with DPBS. Polyclonal goatanti-human FH antiserum is used as a primary antibody (CalBiochem)(diluted 1:100), incubating cells at 4° C. for 15 minutes. Alexa-fluor488-conjugated goat antiserum diluted 1:100 in blocking buffer is usedas the secondary antibody. Cells are examined by flow cytometry(FACScalibur, Becton-Dickinson Immunocytometry, Mountain View, Calif.,USA). Typically, 10,000 events are counted.

Cofactor Activity in Fluid Phase

For the fluid phase cofactor assay, C3b biotin (100 ng/reaction), Factor1 (200 ng/reaction) and 100 ng of purified factor H are used in a totalvolume of 30 μl. Samples taken before and after addition of Factor I areseparated by SDS-PAGE under reducing conditions and analyzed by Westernblotting, detecting and quantitating C3b degradation products byStrepavidin-POD-conjugation (1:10000). C3b (40 μg) (CalBiochem) isbiotinylated using the Biotin Labeling Kit (Roche Diagnostics, Mannheim,Germany), according to the manufacturer's instructions. In brief, 30 μgof C3b (CalBiochem) is labeled with D-biotinyl-epsilon-aminocaproicacid-N-hydroxysuccinimide ester for 2 hours at 25° C. Excess biotin isremoved by gel filtration using a PBS equilibrated PD10 column (AmershamBiosciences). Also see Sanchez-Corral et al., 2002, Am J. Hum. Genet.71:1285-95.

Heparin Binding Assay

Binding of purified CFH proteins (CFH402Y and CFH402H) to heparin isanalyzed using heparin affinity chromatography in a high-performanceliquid chromatograph (HPLC) system. 10 μg of CFH protein is diluted in½×PBS and applied to a heparin-Sepharose affinity column (HiTrap,Amersham Biosciences) at a flow rate of 0.5 ml/min. The column isextensively washed with ½×PBS, and the bound CFH protein eluted using alinear salt gradient ranging from 75 to 500 mM NaCl, in a total volumeof 10 ml and at a flow rate of 0.5 ml/min. Eluted fractions are assayedby SDS-PAGE and Western blot analysis. Elution of isoforms in differentfractions is indicative that specific amino acid variations in the CFHprotein can modulate binding of the protein to heparin. Also see, e.g.,Pangburn et al., 1991, Localization of the heparin-binding site oncomplement Factor H, J Biol Chem. 266:16847-53.

CFHR5 Administration

In another approach, recombinant CFHR5 polypeptide is administered tothe patient. In one embodiment, the recombinant CFHR5 has a neutral-typesequence, or a biologically active fragment thereof. In anotherembodiment the recombinant CFHR5 has the sequence of a protectiveallele, or a protective biologically active fragment thereof. Methodsfor production of therapeutic recombinant proteins are well known andinclude methods described hereinbelow. The therapeutic polypeptide canbe administered systemically (e.g., intravenously or by infusion) orlocally (e.g., directly to an organ or tissue, such as the eye or theliver).

Therapeutic Compositions Containing CFH or CFHR5Polypeptides

The invention provides therapeutic preparations of Factor Hpolypeptides, which may be wild-type or variants (e.g., neutral orprotective variants), and may be full length forms, truncated forms, orbiologically active fragments of the variant Factor H polypeptides. Asdescribed herein, protective Factor H proteins (and genes encoding them)can be identified by identifying an individual as having a protectivehaplotype and determining the amino acid sequence(s) of Factor H encodedin the genome of the individual, where a protective Factor H protein isencoded by an allele having a protective haplotype. Biologically activefragments may include any portion of the full-length Factor Hpolypeptide which confers a biological function on the variant protein.In some cases, a protective haplotype will be associated with expressionof a less-than full length form of Factor H (i.e., in addition to FHL-1)due, for example, to the presence of a premature stop codon in the gene.

Therapeutically active fragments can also be identified by testing theeffect of the protein on expression of AMD biomarkers. Exemplary AMDbiomarkers include complement pathway components (for example, Factor I,Factor H, C1r, C3, C3a), C reactive protein, haptoglobin, apolipoproteinE, immunoglobulin heavy or light chain(s), alpha 1 antitrypsin, alpha 2macroglobulin, transthyretin, creatinine, and others described incopending provisional application No. 60/715,503 entitled “BiomarkersAssociated With Age-Related Macular Degeneration.”

The invention provides therapeutic preparations of CFHR5 polypeptides,which may be wild-type or variants (e.g., neutral or protectivevariants), and may be full length forms or biologically active fragmentsof the variant CFHR5 polypeptides. As described herein, protective CFHR5proteins (and genes encoding them) can be identified by identifying anindividual as having a protective haplotype and determining the aminoacid sequence(s) of CFHR5 encoded in the genome of the individual, wherea protective CFHR5 protein is encoded by an allele having a protectivehaplotype. Biologically active fragments may include any portion of thefull-length CFHR5 polypeptide which confers a biological function on thevariant protein. Therapeutically active fragments can also be identifiedby testing the effect of the protein on expression of AMD biomarkers asdescribed above for Factor H.

Some forms of Factor H and CFHR5 can be isolated from the blood ofgenotyped donors, from cultured or transformed RPE cells derived fromgenotyped ocular donors, or from cell lines (e.g., glial or hepatic)that express endogenous Factor H. Alternatively, therapeutic proteinscan be recombinantly produced (e.g., in cultured bacterial or eukaryoticcells) and purified using methods well known in the art and describedherein. As noted above, some forms of Factor H and CFHR5 have beenrecombinantly expressed for research purposes. However, such researchpreparations are not suitable for therapeutic use. The present inventionprovides recombinant polypeptides suitable for administration topatients including polypeptides that are produced and tested incompliance with the Good Manufacturing Practice (GMP) requirements. Forexample, recombinant polypeptides subject to FDA approval must be testedfor potency and identity, be sterile, be free of extraneous material,and all ingredients in a product (i.e., preservatives, diluents,adjuvants, and the like) must meet standards of purity, quality, and notbe deleterious to the patient.

The invention provides a composition comprising a Factor H polypeptideor CFHR5 polypeptide, and a pharmaceutically acceptable excipient orcarrier. The term “pharmaceutically acceptable excipient or carrier”refers to a medium that is used to prepare a desired dosage form of acompound. A pharmaceutically acceptable excipient or carrier can includeone or more solvents, diluents, or other liquid vehicles; dispersion orsuspension aids; surface active agents; isotonic agents; thickening oremulsifying agents; preservatives; solid binders; lubricants; and thelike. Remington's Pharmaceutical Sciences, Fifteenth Edition, E. W.Martin (Mack Publishing Co., Easton, Pa., 1975) and Handbook ofPharmaceutical Excipients, Third Edition, A. H. Kibbe ed. (AmericanPharmaceutical Assoc. 2000), disclose various carriers used informulating pharmaceutical compositions and known techniques for thepreparation thereof. In one embodiment, the pharmaceutically acceptableexcipient is not deleterious to a mammal (e.g., human patient) ifadministered to the eye (e.g., by intraocular injection). Forintraocular administration, for example and not limitation, thetherapeutic agent can be administered in a Balanced Salt Solution (BSS)or Balanced Salt Solution Plus (BSS Plus) (Alcon Laboratories, FortWorth, Tex., USA). In a related aspect, the invention provides a sterilecontainer, e.g. vial, containing a therapeutically acceptable Factor Hprotein, optionally a lyophilized preparation. Therapeutic Factor Hproteins or CFHR5 polypeptides can be made recombinantly, as describedabove. Alternatively, Factor H protein or CFHR5 polypeptide can beisolated from cultured RPE cells (e.g., primary cultures) or other cellsthat express Factor H or CFHR5 endogenously.

The amount of neutral or protective forms of Factor H or truncatedFactor H, or biologically active fragments thereof, or neutral orprotective forms of CFHR5, or biologically active fragments thereof, tobe administered to an individual can be determined. The normal plasmaconcentration of Factor H varies between 116 and 562 micrograms/ml andthe half-life of Factor H in plasma is about 6½ days (for a recentreview, see Esparza-Gordillo et al., 2004 “Genetic and environmentalfactors influencing the human factor H plasma levels” Immunogenetics56:77-82). In one embodiment, exogenous Factor H can be administered toan individual in an amount sufficient to achieve a level similar to theplasma concentration of Factor H in a healthy individual, i.e., anamount sufficient to achieve a plasma level of from 50 to 600 mg/ml,such as from 100 to 560 mg/ml. The amount of Factor H to be administeredto an individual (e.g., a 160 pound subject) can be, for example and notfor limitation, from 10 milligrams to 5000 milligrams per dose, from 50milligrams to 2000 milligrams per dose, from 100 milligrams to 1500milligrams per dose, from 200 milligrams to 1000 milligrams per dose, orfrom 250 milligrams to 750 milligrams per dose. The frequency with whichFactor H can be administered to an individual can be, for example andnot for limitation, twice per day, once per day, twice per week, onceper week, once every two weeks, once per month, once every two months,once every six months, or once per year. The amount and frequency ofadministration of Factor H to an individual can be readily determined bya physician by monitoring the course of treatment.

B) Gene Therapy Methods

In another approach, Factor H protein or CFHR5 polypeptide isadministered by in vivo expression of protein encoded by exogenouspolynucleotide (i.e., via gene therapy). In one example, gene therapyinvolves introducing into a cell a vector that expresses Factor Hpolypeptide or biologically active fragment or CFHR5 polypeptide orbiologically active fragment.

Vectors can be viral or nonviral. A number of vectors derived fromanimal viruses are available, including those derived from adenovirus,adeno-associated virus, retroviruses, pox viruses, alpha viruses,rhadboviruses, and papillomaviruses. Usually the viruses have beenattenuated to no longer replicate (see, e.g., Kay et al. 2001, NatureMedicine 7:33-40).

The nucleic acid encoding the Factor H polypeptide or CFHR5 polypeptideis typically linked to regulatory elements, such as a promoters and anenhancers, which drive transcription of the DNA in the target cells ofan individual. The promoter may drive expression of the Factor H gene orCFHR5 gene in all cell types. Alternatively, the promoter may driveexpression of the Factor H gene or CFHR5 gene only in specific celltypes, for example, in cells of the retina or the kidney. The regulatoryelements, operably linked to the nucleic acid encoding the Factor Hpolypeptide or CFHR5 polypeptide, are often cloned into a vector.

As will be understood by those of skill in the art, gene therapy vectorscontain the necessary elements for the transcription and translation ofthe inserted coding sequence (and may include, for example, a promoter,an enhancer, other regulatory elements). Promoters can be constitutiveor inducible. Promoters can be selected to target preferential geneexpression in a target tissue, such as the RPE (for recent reviews seeSutanto et al., 2005, “Development and evaluation of the specificity ofa cathepsin D proximal promoter in the eye” Curr Eye Res. 30:53-61;Zhang et al., 2004, “Concurrent enhancement of transcriptional activityand specificity of a retinal pigment epithelial cell-preferentialpromoter” Mol. Vis. 10:208-14; Esumi et al., 2004, “Analysis of the VMD2promoter and implication of E-box binding factors in its regulation” JBiol Chem 279:19064-73; Camacho-Hubner et al., 2000, “The Fugu rubripestyrosinase gene promoter targets transgene expression to pigment cellsin the mouse” Genesis. 28:99-105; and references therein).

Suitable viral vectors include DNA virus vectors (such as adenoviralvectors, adeno-associated virus vectors, lentivirus vectors, andvaccinia virus vectors), and RNA virus vectors (such as retroviralvectors). In one embodiment, an adeno-associated viral (AAV) vector isused. For recent reviews see Auricchio et al., 2005, “Adeno-associatedviral vectors for retinal gene transfer and treatment of retinaldiseases” Curr Gene Ther. 5:339-48; Martin et al., 2004, Gene therapyfor optic nerve disease, Eye 18:1049-55; Ali, 2004, “Prospects for genetherapy” Novartis Found Symp. 255:165-72; Hennig et al., 2004,“AAV-mediated intravitreal gene therapy reduces lysosomal storage in theretinal pigmented epithelium and improves retinal function in adult MPSVII mice” Mol Ther. 10:106-16; Smith et al., 2003, “AAV-Mediated genetransfer slows photoreceptor loss in the RCS rat model of retinitispigmentosa” Mol Ther. 8:188-95; Broderick et al., 2005, “Localadministration of an adeno-associated viral vector expressing IL-10reduces monocyte infiltration and subsequent photoreceptor damage duringexperimental autoimmune uveitis” Mol. Ther. 12:369-73; Cheng et al.,2005, “Efficient gene transfer to retinal pigment epithelium cells withlong-term expression. Retina 25:193-201; Rex et al.,“Adenovirus-mediated delivery of catalase to retinal pigment epithelialcells protects neighboring photoreceptors from photo-oxidative stress.Hum Gene Ther. 15:960-7; and references cited therein).

Gene therapy vectors must be produced in compliance with the GoodManufacturing Practice (GMP) requirements rendering the product suitablefor administration to patients. The present invention provides genetherapy vectors suitable for administration to patients including genetherapy vectors that are produced and tested in compliance with the GMPrequirements. Gene therapy vectors subject to FDA approval must betested for potency and identity, be sterile, be free of extraneousmaterial, and all ingredients in a product (i.e., preservatives,diluents, adjuvants, and the like) must meet standards of purity,quality, and not be deleterious to the patient. For example, the nucleicacid preparation is demonstrated to be mycoplasma-free. See, e.g, Islamet al., 1997, An academic centre for gene therapy research and clinicalgrade manufacturing capability, Ann Med 29, 579-583.

Methods for administering gene therapy vectors are known. In oneembodiment, Factor H or CFHR5 expression vectors are introducedsystemically (e.g., intravenously or by infusion). In one embodiment,Factor H or CFHR5 expression vectors are introduced locally (i.e.,directly to a particular tissue or organ, e.g., liver). In one preferredembodiment, Factor H or CFHR5 expression vectors are introduced directlyinto the eye (e.g., by ocular injection). For recent reviews see, e.g.,Dinculescu et al., 2005, “Adeno-associated virus-vectored gene therapyfor retinal disease” Hum Gene Ther. 16:649-63; Rex et al., 2004,“Adenovirus-mediated delivery of catalase to retinal pigment epithelialcells protects neighboring photoreceptors from photo-oxidative stress”Hum Gene Ther. 15:960-7; Bennett, 2004, “Gene therapy for Lebercongenital amaurosis” Novartis Found Symp. 255:195-202; Hauswirth etal., “Range of retinal diseases potentially treatable by AAV-vectoredgene therapy” Novartis Found Symp. 255:179-188, and references citedtherein).

Thus in one aspect, the invention provides a preparation comprising agene therapy vector encoding a Factor H protein or CFHR5 polypeptide,optionally a viral vector, where the gene therapy vector is suitable foradministration to a human subject and in an excipient suitable foradministration to a human subject (e.g., produced using GLP techniques).Optionally the gene therapy vector comprising a promoter that isexpressed preferentially or specifically in retinal pigmented epitheliumcells.

Nonviral methods for introduction of Factor H or CFHR5 sequences, suchas encapsulation in biodegradable polymers (e.g., polylactic acid (PLA);polyglycolic acid (PGA); and co-polymers (PLGA) can also be used (forrecent reviews see, e.g., Bejjani et al., 2005, “Nanoparticles for genedelivery to retinal pigment epithelial cells” Mol. Vis. 11: 124-32;Mannermaa et al., 2005, “Long-lasting secretion of transgene productfrom differentiated and filter-grown retinal pigment epithelial cellsafter nonviral gene transfer” Curr Eye Res. 2005 30:345-53; andreferences cited therein). Alternatively, the nucleic acid encoding aFactor H polypeptide or CFHR5 polypeptide may be packaged intoliposomes, or the nucleic acid can be delivered to an individual withoutpackaging without using a vector.

C) DNA Repair

In another approach, subjects at risk for developing AMD (and/or withearly stage disease) can have a risk form of Factor H or CFHR5 replacedby a neutral or protective form of Factor H or CFHR5 by DNA repair. Inone embodiment, triplex forming oligonucleotides designed tospecifically bind to polymorphic sites in the Factor H or CFHR5 geneassociated with a risk haplotype can be administered to an individual byviral or nonviral methods. Triplex-forming oligonucleotides bind to themajor groove of duplex DNA in a sequence-specific manner and provoke DNArepair, resulting in the targeted modification of the genome (for arecent review see Kuan et al., 2004, “Targeted gene modification usingtriplex-forming oligonucleotides” Methods Mol. Biol. 262:173-94). Atriplex-forming oligonucleotide that binds to a sequence spanning apolymorphism associated with a risk haplotype provokes DNA repair,resulting in the modification of the sequence from a risk allele to aneutral or protective allele and can ameliorate the development orprogression of disease.

D) Introduction of Cells, Tissues, or Organs Expressing a Neutral orProtective Form of Factor H Protein or CFHR5Polypeptide

In another approach, cells expressing neutral or protective forms ofFactor H or Factor H-Related proteins (e.g., CFHR5) are administered toa patient. In an embodiment the recipient is heterozygous or, moreoften, homozygous for a risk haplotype. For example, hepatocytetransplantation has been used as an alternative to whole-organtransplantation to support many forms of hepatic insufficiency (see,e.g., Ohashi et al., Hepatocyte transplantation: clinical andexperimental application, J Mol Med. 2001 79:617-30). According to thismethod, hepatocytes or other CFH or CFHR5-expressing cells areadministered (e.g., infused) to a patient in need of treatment. Thesecells migrate to the liver or other organ, and produce the therapeuticprotein. Also see, e.g., Alexandrova et al., 2005, “Large-scaleisolation of human hepatocytes for therapeutic application” CellTransplant. 14(10):845-53; Cheong et al., 2004, “Attempted treatment offactor H deficiency by liver transplantation” Pediatr Nephrol. 19:454-8;Ohashi et al., 2001, “Hepatocyte transplantation: clinical andexperimental application” J Mol Med. 79:617-30; Serralta et al., 2005,“Influence of preservation solution on the isolation and culture ofhuman hepatocytes from liver grafts” Cell Transplant. 14(10):837-43;Yokoyama et al., 2006, “In vivo engineering of metabolically activehepatic tissues in a neovascularized subcutaneous cavity” Am. J.Transplant. 6(1):50-9; Dhawan et al., 2005, “Hepatocyte transplantationfor metabolic disorders, experience at King's College hospital andreview of literature.” Acta Grastroenterol. Belg. 68(4):457-60; Bruns etal., 2005, “Injectable liver: a novel approach using fibrin gel as amatrix for culture and intrahepatic transplantation of hepatocytes”Tissue Eng. 11(11-12): 1718-26. Other cell types that may be usedinclude, for illustration and not limitation, kidney and pancreaticcells. In one embodiment, the administered cells are engineered toexpress a recombinant form of the protein.

In another, related approach, therapeutic organ transplantation is used.Most of the body's systemic Factor H is produced by the liver, makingtransplantation of liver tissue the preferred method. See, Gerber etal., 2003, “Successful (?) therapy of hemolytic-uremic syndrome withfactor H abnormality” Pediatr Nephrol. 18:952-5.

In another approach, a protective form of CFH protein is delivered tothe back of the eye by injection into the eye (e.g. intravitreal) or viaencapsulated cells. Neurotech's Encapsulated Cell Technology (ECT), asan example, is a unique technology that allows for the sustained,longterm delivery of therapeutic factors to the back of the eye. See(http://www.neurotech.fr). ECT implants consist of cells that have beengenetically modified to produce a specific therapeutic protein that areencapsulated in a semi-permeable hollow fiber membrane. The cellscontinuously produce the therapeutic protein that diffuses out of theimplant and into the eye (Bush et al 2004). CNTF delivered to the humaneye by ECT devices was recently shown to be completely successful andassociated with minimal complications in 10 patients enrolled in a PhaseI clinical trial (Sieving et al 2005). Also see Song et al., 2003; Tao2002., and Hammang et al., U.S. Pat. No. 6,649,184. Inh one embodimentof the present invention, a protective form of Factor H (including theso-called neutral form) is expressed in cells and administered in anencapsulated form. In one embodiment, the cells used are the NTC-201human RPE line (ATCC # CRL-2302) available from the American TypeCulture Collection P.O. Box 1549, Manassas, Va. 20108.

E) Therapy to Decrease Levels of Risk Variant of Factor H or CFHR5

Loss of the normal or protective function of Factor H or CFHR5 may beassociated with AMD. Non-synonymous polymorphisms in the Factor H andCFHR5 genes, such as those shown in TABLES 1A, 1B, 1C, 11, 14 and 15,showing the strongest correlation with AMD and resulting in a variantFactor H polypeptide or CFHR5 polypeptide, are likely to have acausative role in AMD. For example, the variant Factor H or CFHR5 mayact as a so-called “dominant-negative” mutant interfering with normalFactor H or CFHR5 function.

Any method of reducing levels of the risk forms of Factor H or CFHR5 inthe eye or systemically may be used for treatment including, forexample, inhibiting transcription of a Factor H or CFHR5 gene,inhibiting translation of Factor H or CFHR5 RNA, increasing the amountor activity of a neutral or protective form of Factor H or truncatedFactor H, or biologically active fragment thereof, increasing the amountor activity of a neutral or protective form of CFHR5 polypeptide, orbiologically active fragment thereof, or decreasing the amount oractivity of Factor H protein or CFHR5 polypeptides (e.g., byplasmaphoresis, antibody-directed plasmaphoresis, or complexing with aFactor H or CFHR5 binding moiety, e.g., heparin or variant specificantibody). In some embodiments levels of Factor H or CFHR5 arepreferentially reduced in the eye (e.g., RPE) relative to other tissues.For illustration and not limitation, several methods are brieflydescribed below.

In one approach, a subject identified as being at risk for AMD istreated by administration of heparin. Heparin and heparin derivatives(including heparinoids) may have promising therapeutic properties forthe treatment various complement-associated diseases, including MPGNII(Floege et al., 1993; Girardi, 2005; Diamond and Karnovsky, 1986;Striker, 1999; Rops et al., 2004). In view of the association betweenAMD and MPGNII disclosed herein, heparin and heparin derivatives(including heparinoids) may be efficacious for the treatment of AMD. Ina clinical trial of patients with chronic proliferativeglomerulonephritis receiving daily subcutaneous injections of heparinfor over one year, Cade and colleagues reported improved creatinineclearance and a regression of glomerular hypercellularity (Cade et al.,1971). Both heparin and low molecular weight heparin (Enoxaparin) havebeen shown to prevent the progression of antiphospholipid antibodysyndrome in mice by blocking the alternative and classical pathways ofthe complement cascade (Girardi et al., 2004). The anti-complementactivity of heparin includes blockade of the formation of C3bBb, theamplification convertase by the alternative pathway; fluid phase heparinprevents the generation of C3bBb by inhibiting the interaction of C3bwith factor B and factor D (Weiler et al., 1976).

F) Administration of Inhibitory Nucleic Acids

Antisense nucleic acids—Antisense nucleic acids, such as purifiedanti-sense RNA complementary to the RNA encoding a variant Factor Hpolypeptide can be used to inhibit expression of a Factor H geneassociated with a risk haplotype. For recent reviews see, e.g., Gomes etal., 2005, “Intraocular delivery of oligonucleotides” Curr PharmBiotechnol. 6:7-15; and Henry et al., 2004, “Setting sights on thetreatment of ocular angiogenesis using antisense oligonucleotides”Trends Pharmacol Sci 25:523-7; and references cited therein.

RNA Interference—Double stranded RNA (dsRNA) inhibition methods can alsobe used to inhibit expression of HF1. The RNA used in such methods isdesigned such that at least a region of the dsRNA is substantiallyidentical to a region of the HF1 gene; in some instances, the region is100% identical to the HF1 gene. For use in mammals, the dsRNA istypically about 19-30 nucleotides in length (i.e., short interferingRNAs are used (siRNA or RNAi)), and most often about 21 nucleotides inlength. Methods and compositions useful for performing dsRNAi and siRNAare discussed, for example, in PCT Publications WO 98/53083; WO99/32619; WO 99/53050; WO 00/44914; WO 01/36646; WO 01/75164; WO02/44321; and U.S. Pat. No. 6,107,094. siRNA can be is synthesized invitro and administered to a patient. Alternatively, RNAi strategies canbe successfully combined with vector-based approaches to achievesynthesis in transfected cells of small RNAs from a DNA template (see,e.g., Sui et al., 2002, “A DNA vector-based RNAi technology to suppressgene expression in mammalian cells” Proc Natl Acad Sci USA 99:5515-20;and Kasahara and Aoki, 2005, “Gene silencing using adenoviral RNAivector in vascular smooth muscle cells and cardiomyocytes” Methods Mol.Med. 112:155-72; and references cited therein).

Ribozymes—Ribozymes are enzymatic RNA molecules capable of catalyzingthe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Withinthe scope of the invention are engineered hammerhead motif ribozymemolecules that can specifically and efficiently catalyze endonucleolyticcleavage of the sequence encoding human Factor H. Specific ribozymecleavage sites within any potential RNA target are initially identifiedby scanning the target molecule for ribozyme cleavage sites whichinclude the sequences such as, GUA, GUU and GUC. Once identified, shortRNA sequences of between 15 and 20 ribonucleotides corresponding to theregion of the target gene containing the cleavage site may be evaluatedfor secondary structural features which may render the oligonucleotideinoperable. The suitability of candidate targets may also be evaluatedby testing accessibility to hybridization with complementaryoligonucleotides using ribonuclease protection assays. Properties ofribozymes are well known in the art; for a general description seepatents by Cech (U.S. Pat. No. 6,180,399; U.S. Pat. No. 5,869,254; U.S.Pat. No. 6,025,167; U.S. Pat. No. 5,854,038; U.S. Pat. No. 5,591,610;U.S. Pat. No. 5,667,969; U.S. Pat. No. 5,354,855;U.S. Pat. No.5,093,246; U.S. Pat. No. 5,180,818; U.S. Pat. No. 5,116,742; U.S. Pat.No. 5,037,746; and U.S. Pat. No. 4,987,071). Ribozymes and otherinhibitory nucleic acids can be designed to preferentially inhibitexpression of a gene having a sequence associated with a risk haplotype.Thus, a ribozyme that recognizes the sequence spanning the polymorphismand cleaving adjacent to GUA recognizes the risk form but not theneutral or protective form, allowing selective cleavage (Dawson et al.,2000, “Hammerhead ribozymes selectively suppress mutant type I collagenmRNA in osteogenesis imperfecta fibroblasts” Nucleic Acids Res.28:4013-20; Blalock et al., 2004 “Hammerhead ribozyme targetingconnective tissue growth factor mRNA blocks transforming growthfactor-beta mediated cell proliferation” Exp Eye Res. 78:1127-36).

Triplex-Forming Oligonucleotides—Triplex-forming oligonucleotides bindto the major groove of duplex DNA in a sequence-specific manner andprovoke DNA repair, resulting in the targeted modification of the genome(for a recent review see Kuan et al., 2004, “Targeted gene modificationusing triplex-forming oligonucleotides” Methods Mol. Biol. 262:173-94).Oligonucleotides can be designed to specifically bind to polymorphicsites in the Factor H gene associated with a risk haplotype. Atriplex-forming oligonucleotide that binds to a sequence spanning apolymorphism associated with a risk haplotype provokes DNA repair,resulting in the modification of the sequence from a risk allele to aneutral or protective allele.

Similar antisense nucleic acid, RNA interference, ribozyme andtriplex-forming pliognucleotide methodologies as described above may beused to reduce levels of risk forms of CFHR5 in the eye or systemicallyfor treatment of AMD.

It will be understood that inhibitory nucleic acids can be administeredas a pharmaceutical composition or using gene therapy methods.

G) Antibody Therapy

In one aspect, an anti-HF1 antibody that specifically interacts with andneutralizes the activity of a variant Factor H polypeptide isadministered to an individual with or at risk for AMD. In oneembodiment, the antibody recognizes both wild-type and variant Factor Hprotein. In one embodiment, the antibody recognizes the variant but notthe wild-type Factor H protein. In another aspect, an anti-CFHR5antibody that specifically interacts with and neutralizes the activityof a variant CFHR5 polypeptide is administered to an individual with orat risk for AMD. In one embodiment, the antibody recognizes bothwild-type and variant CFHR5 protein. In one embodiment, the antibodyrecognizes the variant but not the wild-type CFHR5 protein. The antibodycan be administered systemically or locally (see, e.g., Gaudreault etal., 2005, “Preclinical pharmacokinetics of Ranibizumab (rhuFabV2) aftera single intravitreal administration” Invest Ophthalmol Vis Sci.46:726-33). Methods for making anti-HF1 and anti-CFHR5 antibodies areknown in the art, and include methods described below. In a relatedaspect, an agent that preferentially interacts with and reduces theactivity of a variant Factor H polypeptide and/or CFHR5 polypeptide isadministered to an individual with or at risk for AMD.

H) Modulators of the Alternative Pathway

In one aspect the invention provides methods for treating AMD byadministering an agent (e.g., native protein, recombinant protein,antibody, or small molecule) directed at modulating the alternativepathway (AP) of the complement cascade, either locally in the eye or atthe systemic level. In one embodiment, the treatment comprisesadministering an agent that modulates the AP directly. In oneembodiment, the treatment comprises administering an agent thatmodulates the triggering of the AP (e.g., microbes). In one embodiment,the treatment comprises administering an agent that modulates pathwaysdownstream from the AP. Exemplary agents that modulate the AP are knownin the art and include, but are not limited to, DFP, PR226, BCX-1470,FUT-175, sMCP, PS-oligo, Compstatin, Fucan, and GCRF (see, e.g.,Makrides, 1998, “Therapeutic inhibition of the complement system”Pharmacol Rev. 50:59-87; Holland et al., 2004, “Synthetic small moleculecomplement inhibitors” Curr Opin Investig Drugs 5:1163-73; Holers etal., 2004, “The alternative pathway of complement in disease:opportunities for therapeutic targeting” Mol Immunol. 41:147-52). APmodulators can be administered systemically or by intraocular injectionor other methods known for delivery of compounds to the eye.

I) Drug Screening/Antagonists of Risk Variant Factor H or Variant CFHR5

The invention provides a method of screening for an agent effective fortreatment of AMD by contacting a variant protein, host cell ortransgenic animal expressing a Factor H or CFHR5 variant, and monitoringbinding, expression, processing or activity of the variant. In anembodiment, the Factor H variant has valine at amino acid 62 and/or hashistidine at amino acid 402 and/or has cysteine at amino acid 1210. Inan embodiment, the CFHR5 variant has a serine at amino acid 46.

Antagonists of variant Factor H polypeptides (e.g., variants associatedwith risk haplotypes) can be used to treat AMD. Antagonists may suppressexpression of variant Factor H, suppress activity, or reduce RNA orprotein stability. Antagonists can be obtained by producing andscreening large combinatorial libraries, which can be produced for manytypes of compounds in a step-wise and high throughput fashion. Suchcompounds include peptides, polypeptides, beta-turn mimetics,polysaccharides, phospholipids, hormones, prostaglandins, steroids,aromatic compounds, heterocyclic compounds, benzodiazepines, oligomericN-substituted glycines and oligocarbamates, and the like. Largecombinatorial libraries of the compounds can be constructed by methodsknown in the art. See e.g., WO 95/12608; WO 93/06121; WO 94/08051; WO95/35503; WO 95/30642 and WO 91/18980. Libraries of compounds areinitially screened for specific binding to the variant Factor Hpolypeptide. Compounds with in vitro binding activity can also beassayed for their ability to interfere with a biological activity of thevariant Factor H polypeptide, for example, binding to C3b or to heparin.Antagonist activity can be assayed in either a cell-based system or in atransgenic animal model in which exogenous variant Factor H polypeptideis expressed.

Antagonists of variant CFHR5 polypeptides (e.g., variants associatedwith risk haplotypes) can be used treat AMD and can be obtained asdescribed above for variant Factor H antagonists.

J) Patient Specific Therapy

Customized therapies can be devised for groups of patients withdifferent genetic subtypes of AMD, based upon the presence of certainpolymorphisms in the Factor H gene or CFHR5 gene having causative rolesin AMD and having elucidated the effect of these polymorphisms on theexpression level and/or biological activity of variant Factor Hpolypeptides or CFHR5 polypeptides. For example, if a polymorphism inFactor H or CFHR5 causes AMD in an animal model by increasing theexpression level and/or biological activity of a variant Factor Hpolypeptide or CFHR5 polypeptide, AMD associated with the Factor H orCFHR5 polymorphism can be treated by administering to a patient anantagonist of the variant Factor H polypeptide or variant CFHR5polypeptide.

K) Assessing Therapeutic Efficacy Using AMD Biomarkers

As noted above, therapeutic efficacy of particular fragments of CFH orCFHR proteins can also be determined by testing the effect of theprotein on expression of AMD biomarkers. Exemplary AMD biomarkersinclude those described hereinabove. These AMD-associated proteins(biomarkers) are present in individuals with AMD at different (elevatedor reduced) levels compared to healthy individuals. The inventionprovides methods of assessing the efficacy of treatment of AMD andmonitoring the progression of AMD by determining a level of abiomarker(s) in an individual with AMD being treated for the disease andcomparing the level of the biomarker(s) to an earlier determined levelor a reference level of the biomarker. As described in copendingprovisional application No. 60/715,503. the level of the biomarker(s)can be determined by any suitable method, such as conventionaltechniques known in the art, including, for example and not forlimitation, separation-based methods (e.g., gel electrophoresis),immunoassay methods (e.g., antibody-based detection) and function-basedmethods (e.g., enzymatic or binding activity). In one embodiment, amethod of assessing the efficacy of treatment of AMD in a individualinvolves obtaining a sample from the individual and determining thelevel of the biomarker(s) by separating proteins by 2-dimensionaldifference gel electrophoresis (DIGE).

VIII. Factor H and CFHR5 Nucleic Acids

A) Primers and Probes

The invention provides nucleic acids adjacent to or spanning thepolymorphic sites. The nucleic acids can be used as probes or primers(including Invader, Molecular Beacon and other fluorescence resonanceenergy transfer (FRET) type probes) for detecting Factor Hpolymorphisms. In one embodiment, the probes or primers recognize theinsertion in intron 2 but do not recognize the wild-type sequence.Exemplary nucleic acids comprise sequences that span at least one of thepolymorphisms listed in TABLES 1A, 1B, 1C, 11, 14 and 15 in which thepolymorphic position is occupied by an alternative base for thatposition. The base for that position, which is found more frequently inthe control population, is denoted the normal or wild-type sequence,whereas the alternative base for that position, which is found lessfrequently in the control population, is denoted the variant sequence.The nucleic acids also comprise sequences that span other polymorphismsknown in the Factor H and CFHR5 genes, such as polymorphisms identifiedin Tables A and B above.

B) Expression Vectors and Recombinant Production of Factor H and CFHR5Polypeptides.

The invention provides vectors comprising nucleic acid encoding theFactor H polypeptide. The Factor H polypeptide may be wild-type or avariant (e.g., a protective variant) and may be a full-length form(e.g., HF1) or a truncated form. The nucleic acid may be DNA or RNA andmay be single-stranded or double-stranded.

Some nucleic acids encode full-length, variant forms of Factor Hpolypeptides. The variant Factor H polypeptide may differ from normal orwild-type Factor H at an amino acid encoded by a codon including one ofany non-synonymous polymorphic position known in the Factor H gene. Inone embodiment, the variant Factor H polypeptides differ from normal orwild-type Factor H polypeptides at an amino acid encoded by a codonincluding one of the non-synonymous polymorphic positions shown in TABLE1A, TABLE 1B and/or TABLE 1C, that position being occupied by the aminoacid shown in TABLE 1A, TABLE 1B and/or TABLE 1C. It is understood thatvariant Factor H genes may be generated that encode variant Factor Hpolypeptides that have alternate amino acids at multiple polymorphicsites in the Factor H gene.

The invention provides vectors comprising nucleic acid encoding theCFHR5 polypeptide. The CFHR5 polypeptide may be wild-type or a variant(e.g., a protective variant). The nucleic acid may be DNA or RNA and maybe single-stranded or double-stranded.

Some nucleic acids encode full-length, variant forms of CFHR5polypeptides. The variant CFHR5 polypeptide may differ from normal orwild-type CFHR5 at an amino acid encoded by a codon including one of anynon-synonymous polymorphic position known in the CFHR5 gene. In oneembodiment, the variant CFHR5 polypeptides differ from normal orwild-type CFHR5 polypeptides at an amino acid encoded by a codonincluding one of the non-synonymous polymorphic positions shown inTABLES 14 and 15, that position being occupied by the amino acid shownin TABLES 14 and 15. It is understood that variant CFHR5 genes may begenerated that encode variant CFHR5 polypeptides that have alternateamino acids at multiple polymorphic sites in the CFHR5 gene.

Expression vectors for production of recombinant proteins and peptidesare well known (see Ausubel et al., 2004, Current Protocols In MolecularBiology, Greene Publishing and Wiley-Interscience, New York). Suchexpression vectors include the nucleic acid sequence encoding the FactorH polypeptide linked to regulatory elements, such a promoter, whichdrive transcription of the DNA and are adapted for expression inprokaryotic (e.g., E. coli) and eukaryotic (e.g., yeast, insect ormammalian cells) hosts. A variant Factor H or CFHR5 polypeptide can beexpressed in an expression vector in which a variant Factor H or CFHR5gene is operably linked to a promoter. Usually, the promoter is aeukaryotic promoter for expression in a mammalian cell. Usually,transcription regulatory sequences comprise a heterologous promoter andoptionally an enhancer, which is recognized by the host cell.Commercially available expression vectors can be used. Expressionvectors can include host-recognized replication systems, amplifiablegenes, selectable markers, host sequences useful for insertion into thehost genome, and the like.

Suitable host cells include bacteria such as E. coli, yeast, filamentousfungi, insect cells, and mammalian cells, which are typicallyimmortalized, including mouse, hamster, human, and monkey cell lines,and derivatives thereof. Host cells may be able to process the variantFactor H or CFHR5 gene product to produce an appropriately processed,mature polypeptide. Such processing may include glycosylation,ubiquitination, disulfide bond formation, and the like.

Expression constructs containing a variant Factor H or CFHR5 gene areintroduced into a host cell, depending upon the particular constructionand the target host. Appropriate methods and host cells, both procaryticand eukaryotic, are well-known in the art. Recombinant full-length humanFactor H has been expressed for research purposes in Sf9 insect cells(see Sharma and Pangburn, 1994, Biologically active recombinant humancomplement factor H: synthesis and secretion by the baculovirus system,Gene 143:301-2). Recombinant fragments of human Factor H have beenexpressed for research purposes in a variety of cell types (see, e.g.,Cheng et al., 2005, “Complement factor H as a marker for detection ofbladder cancer” Clin Chem. 5:856-63; Vaziri-Sani et al., 2005, “Factor Hbinds to washed human platelets” J Thromb Haemost. 3:154-62; Gordon etal., 1995, “Identification of complement regulatory domains in humanfactor H” J Immunol. 155:348-56). Recombinant full-length human CFHR5has been expressed for research purposes in Sf9 insect cells (see McRaeet al., 2001, Human Factor H-related Protein 5 (FHR-5), J. Biol. Chem.276:6747-6754).

A variant Factor H or CFHR5 polypeptide may be isolated by conventionalmeans of protein biochemistry and purification to obtain a substantiallypure product. For general methods see Jacoby, Methods in EnzymologyVolume 104, Academic Press, New York (1984); Scopes, ProteinPurification, Principles and Practice, 2nd Edition, Springer-Verlag, NewYork (1987); and Deutscher (ed) Guide to Protein Purification, Methodsin Enzymology, Vol. 182 (1990). Secreted proteins, like Factor H orCFHR5, can be isolated from the medium in which the host cell iscultured. If the variant Factor H or CFHR5 polypeptide is not secreted,it can be isolated from a cell lysate.

In one embodiment the vector is an expression vector for production of avariant Factor H protein having a sequence having non-wildtype sequenceat one or more of the polymorphic sites shown in TABLES 1A, 1B and/orIC.

In one embodiment the vector is an expression vector for production of avariant Factor H protein having a sequence of a protective variant ofFactor H.

In one embodiment the vector is an expression vector for production of avariant CFHR5 protein having a sequence having non-wildtype sequence atone or more of the polymorphic sites shown in TABLES 14 and 15.

In one embodiment the vector is an expression vector for production of avariant CFHR5 protein having a sequence of a protective variant ofFactor H.

C) Gene Therapy Vectors

Methods for expression of Factor H polypeptides or CFHR5 polypeptidesfor gene therapy are known and are described in Section IV(A) above.

XI. Antibodies

The invention provides Factor H-specific antibodies that may recognizethe normal or wild-type Factor H polypeptide or a variant Factor Hpolypeptide in which one or more non-synonymous single nucleotidepolymorphisms (SNPs) are present in the Factor H coding region. In oneembodiment, the invention provides antibodies that specificallyrecognize variant Factor H polypeptides or fragments thereof, but notFactor H polypeptides not having a variation at the polymorphic site.

The invention also provides CFHR5-specific antibodies that may recognizethe normal or wild-type CFHR5 polypeptide or a variant CFHR5 polypeptidein which one or more non-synonymous single nucleotide polymorphisms(SNPs) are present in the CFHR5 coding region. In one embodiment, theinvention provides antibodies that specifically recognize variant CFHR5polypeptides or fragments thereof, but not CFHR5 polypeptides not havinga variation at the polymorphic site.

The antibodies can be polyclonal or monoclonal, and are made accordingto standard protocols. Antibodies can be made by injecting a suitableanimal with a variant Factor H or variant CFHR5 polypeptide, or fragmentthereof, or synthetic peptide fragments thereof. Monoclonal antibodiesare screened according to standard protocols (Koehler and Milstein 1975,Nature 256:495; Dower et al., WO 91/17271 and McCafferty et al., WO92/01047; and Vaughan et al., 1996, Nature Biotechnology, 14: 309; andreferences provided below). In one embodiment, monoclonal antibodies areassayed for specific immunoreactivity with the variant Factor H or CFHR5polypeptide, but not the corresponding wild-type Factor H or CFHR5polypeptide, respectively. Methods to identify antibodies thatspecifically bind to a variant polypeptide, but not to the correspondingwild-type polypeptide, are well-known in the art. For methods, includingantibody screening and subtraction methods; see Harlow & Lane,Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York(1988); Current Protocols in Immunology (J. E. Coligan et al., eds.,1999, including supplements through 2005); Goding, MonoclonalAntibodies, Principles and Practice (2d ed.) Academic Press, New York(1986); Burioni et al., 1998, “A new subtraction technique for molecularcloning of rare antiviral antibody specificities from phage displaylibraries” Res Virol. 149(5):327-30; Ames et al., 1994, Isolation ofneutralizing anti-C5a monoclonal antibodies from a filamentous phagemonovalent Fab display library. J Immunol. 152(9):4572-81; Shinohara etal., 2002, Isolation of monoclonal antibodies recognizing rare anddominant epitopes in plant vascular cell walls by phage displaysubtraction. J Immunol Methods 264(1-2):187-94. Immunization orscreening can be directed against a full-length variant protein or,alternatively (and often more conveniently), against a peptide orpolypeptide fragment comprising an epitope known to differ between thevariant and wild-type forms. Particular variants include the Y402H or162V variants of CFH and HFL1, tha R1210C variant of CFH, the P46Svariant of CFHR5, and truncated forms of CFH. In one embodiment the HF1is measured. As discussed above, in one embodiment the ratio of HFL1 andCHF is measured. Monoclonal antibodies specific for variant Factor H orCFHR5 polypeptides (i.e., which do not bind wild-type proteins, or bindat a lower affinity) are useful in diagnostic assays for detection ofthe variant forms of Factor H or CFHR5, or as an active ingredient in apharmaceutical composition.

The present invention provides recombinant polypeptides suitable foradministration to patients including antibodies that are produced andtested in compliance with the Good Manufacturing Practice (GMP)requirements. For example, recombinant antibodies subject to FDAapproval must be tested for potency and identity, be sterile, be free ofextraneous material, and all ingredients in a product (i.e.,preservatives, diluents, adjuvants, and the like) must meet standards ofpurity, quality, and not be deleterious to the patient.

The invention provides a composition comprising an antibody thatspecifically recognizes a Factor H or CFHR5 polypeptide (e.g., a normalor wild-type Factor H polypeptide or a variant Factor H polypeptide, ora normal or wild-type CFHR5 polypeptide or a variant CFHR5 polypeptide)and a pharmaceutically acceptable excipient or carrier.

In a related aspect, the invention provides a sterile container, e.g.vial, containing a therapeutically acceptable Factor H-specific orCFHR5-specific antibody. In one embodiment it is a lyophilizedpreparation.

In a related aspect, the invention provides pharmaceutical preparationsof human or humanized anti-Factor H or anti-CFHR5 antibodies foradministration to patients. Humanized antibodies have variable regionframework residues substantially from a human antibody (termed anacceptor antibody) and complementarity determining regions substantiallyfrom a mouse-antibody, (referred to as the donor immunoglobulin). See,Peterson, 2005, Advances in monoclonal antibody technology: geneticengineering of mice, cells, and immunoglobulins, ILAR J. 46:314-9,Kashmiri et al., 2005, SDR grafting—a new approach to antibodyhumanization, Methods 356:25-34, Queen et al., Proc. Natl: Acad. Sci.USA 86:10029-10033 (1989), WO 90/07861, U.S. Pat. No. 5,693,762, U.S.Pat. No. 5,693,761, U.S. Pat. No. 5,585,089, U.S. Pat. No. 5,530,101,and Winter, U.S. Pat. No. 5,225,539. The constant region(s), if present,are also substantially or entirely from a human immunoglobulin. Thehuman variable domains are usually chosen from human antibodies whoseframework sequences exhibit a high degree of sequence identity with themurine variable region domains from which the CDRs were derived. Theheavy and light chain variable region framework residues can be derivedfrom the same or different human antibody sequences. The human antibodysequences can be the sequences of naturally occurring human antibodiesor can be consensus sequences of several human antibodies. See Carter etal., WO 92/22653. Certain amino acids from the human variable regionframework residues are selected for substitution based on their possibleinfluence on CDR conformation and/or binding to antigen. Investigationof such possible influences is by modeling, examination of thecharacteristics of the amino acids at particular locations, or empiricalobservation of the effects of substitution or mutagenesis of particularamino acids.

For example, when an amino acid differs between a murine variable regionframework residue and a selected human variable region frameworkresidue, the human framework amino acid should usually be substituted bythe equivalent framework amino acid from the mouse antibody when it isreasonably expected that the amino acid: (1) noncovalently binds antigendirectly, (2) is adjacent to a CDR region, (3) otherwise interacts witha CDR region (e.g. is within about 6A of a CDR region), or (4)participates in the VL-VH interface.

Other candidates for substitution are acceptor human framework aminoacids that are unusual for a human immunoglobulin at that position.These amino acids can be substituted with amino acids from theequivalent position of the mouse donor antibody or from the equivalentpositions of more typical human immunoglobulins. Other candidates forsubstitution are acceptor human framework amino acids that are unusualfor a human immunoglobulin at that position. The variable regionframeworks of humanized immunoglobulins usually show at least 85%sequence identity to a human variable region framework sequence orconsensus of such sequences.

IX. Identification of Risk, Protective, and Neutral Variations andHaplotypes

The invention provides methods of screening for polymorphic sites linkedto polymorphic sites in the Factor H gene and/or CFHR5 gene described inTABLES IA, 1B, 1C, 11, 14 and 15. These methods involve identifying apolymorphic site in a gene that is linked to a polymorphic site in theFactor H gene or CFHR5 gene, wherein the polymorphic form of thepolymorphic site in the Factor H gene or CFHR5 gene is associated AMD(e.g., increased or decreased risk), and determining haplotypes in apopulation of individuals to indicate whether the linked polymorphicsite has a polymorphic form in equilibrium or disequilibrium with thepolymorphic form of the Factor H gene or CFHR5 gene that correlates withthe AMD phenotype.

Polymorphisms in the Factor H gene or CFHR5 gene, such as those shown inTABLES 1A, 1B, 1C, 11, 14 and 15, can be used to establish physicallinkage between a genetic locus associated with a trait of interest andpolymorphic markers that are not associated with the trait, but are inphysical proximity with the genetic locus responsible for the trait andco-segregate with it. Mapping a genetic locus associated with a trait ofinterest facilitates cloning the gene(s) responsible for the traitfollowing procedures that are well-known in the art.

Polymorphisms in the Factor H gene or CFHR5 gene, such as those shown inTABLES 1A, 1B, 1C, 11, 14 and 15, can be used in familial linkagestudies to determine which polymorphisms co-segregate with a phenotypictrait, to determine individuals who require therapy, and to determinethe effects of therapy.

Linkage is analyzed by calculation of a LOD (log of the odds) score,which is the log₁₀ of the ratio of the likelihood of obtaining observedsegregation data for a marker and a genetic locus when the two arelocated at a recombination fraction theta, versus the situation in whichthe two are not linked (segregating independently). See Thompson &Thompson, Genetics in Medicine (5th ed, W.B. Saunders Company,Philadelphia, 1991) and Strachan, “Mapping the human genome” in TheHuman Genome (BIOS Scientific Publishers Ltd, Oxford) Chapter 4). A LODscore of 3 indicates a 1000 to 1 odds against an apparent observedlinkage being a coincidence. A LOD score of +3 or greater is considereddefinitive evidence that two loci are linked, whereas LOD score of −2 orless is considered definitive evidence against linkage.

X. Transgenic Non-Human Animals

The invention provides transgenic non-human animals capable ofexpressing human variant Factor H or CFHR5 polypeptides. Transgenicnon-human animals may have one or both of alleles of the endogenousFactor H or CFHR5 gene inactivated. Expression of an exogenous variantFactor H or CFHR5 gene is usually achieved by operably linking the geneto a promoter and optionally an enhancer, and then microinjecting theconstruct into a zygote following standard protocols. See Hogan et al.,“Manipulating the Mouse Embryo, A Laboratory Manual,” Cold Spring HarborLaboratory. The endogenous Factor H or CFHR5 genes can be inactivated bymethods known in the art (Capecchi, 1989). Factor H deficient mice areavailable for the introduction of exogenous human variant Factor Hgenes. Transgenic animals expressing human or non-human variant Factor Hor CFHR5 polypeptides provide useful drug screening systems and asmodels of AMD and other complement related diseases. Transgenic animalsmay also be used for production of recombinant CFH and CFHR5 proteins ofthe invention (see, e.g. U.S. Pat. Nos. 6,066,725; 6,013,857; 5,994,616;and 5,959,171; Lillico et al., 2005; Houdebine, 2000).

XI. Kits

The invention provides reagents, devices and kits detecting Factor H orCFHR5 polymorphisms and haplotypes. Although particularly suited forscreening for risk of developing AMD and/or for identifying appropriatetherapy for preventing or ameliorating AMD in a subject, it will beunderstood that in certain embodiments these reagents, devices and kitscan be used for analysis of Factor H and CFHR5 polymorphisms andhaplotypes for any purpose, including but not limited to determiningrisk of developing MPGNII or any other complement associated condition.

A number of assay systems are known in the art, and it is within theskill of the art to arrive at means to determine the presence ofvariations associated with AMD. The kit reagents, such as multipleprimers, multiple probes, combinations of primers, or combinations ofprobes, may be contained in separate containers prior to their use fordiagnosis or screening. In an embodiment, the kit contains a firstcontainer containing a probe, primer, or primer pair for a first CFH orCFHR5 allele described herein, and a second container containing aprobe, primer, or primer pair for a second CFH or CFHR5 allele describedherein.

In one embodiment, the invention provides kits comprising at least oneFactor H or CFHR5 allele-specific oligonucleotide that hybridizes to aspecific polymorphism in the Factor H or CFHR5 gene. The kits maycontain one or more pairs of Factor H or CFHR5 allele-specificoligonucleotides hybridizing to different forms of a polymorphism. TheFactor H or CFHR5 allele-specific oligonucleotides may include sequencesderived from the coding (exons) or non-coding (promoter, 5′untranslated, introns or 3′ untranslated) region of the Factor H orCFHR5 gene. The Factor H or CFHR5 allele-specific oligonucleotides maybe provided immobilized on a substrate. The substrate may compriseFactor H or CFHR5 allele-specific oligonucleotide probes for detectingat least 2, 3, 4, 5, more than 5, (e.g., at least 6, 7, or 8) or all ofthe polymorphisms shown in TABLES 1A, 1B, 1C, 11, 14 and 15 and/or otherpolymorphisms in the Factor H or CFHR5 gene (e.g., includingpolymorphisms listed above that are found in the SNP database). In oneembodiment the kit is used to diagnose AMD. In a related embodiment, thekit is used to screen for another disease associated with variation inthe Factor H or CFHR5 gene.

The kit may include at least one Factor H- or CFHR5-specific primer thathybridizes spanning or adjacent to a specific polymorphism in the FactorH or CFHR5 gene. The Factor H- or CFHR5-specific primers may includesequences derived from the coding (exons) or non-coding (promoter, 5′untranslated, introns or 3′ untranslated) region of the Factor H orCFHR5 gene. Often, the kits contain one or more pairs of Factor H- orCFHR5-specific primers that hybridize to opposite strands of nucleicacid adjacent to a specific polymorphism in the Factor H or CFHR5 gene.In the presence of appropriate buffers and enzymes, the Factor H- orCFHR5-specific primer pairs are useful in amplifying specificpolymorphisms in the Factor H or CFHR5 gene.

It will be apparent to the skilled practitioner guided by thisdisclosure that various polymorphisms and haplotypes can be detected toassess the propensity of an individual to develop a Factor H relatedcondition. The following examples and combinations are provided forillustration and not limitation. In some cases, the assay identifies theallele at least one, at least two, at least three, at least four, atleast five or at least six polymorphic sites in the Factor H or CFHR5gene. In some cases, the assay identifies the allele at 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or all of the polymorphisms in the Factor H or CFHR5gene listed in TABLES 1A, 1B, 1C, 11, 14 and 15. In one embodiment, thesites are selected from: rs529825; rs800292; rs3766404; rs1061147;rs1061170; rs203674; and optionally including exon 22 (R120C). In oneembodiment, the sites are selected from rs529825; rs800292; intron 2(IVS2 or insTT); rs3766404; rs1061147; rs1061170; exon 10A; rs203674;rs375046; and optionally including exon 22 (R120C). In one embodiment,the sites are selected from: rs3753394; rs529825; rs800292; intron 2(IVS2 or insTT); rs3766404; rs1061147; rs1061170; rs2274700; rs203674;rs3753396; rs1065489; and optionally including exon 22 (R1210C). In oneembodiment, the sites are selected from: rs800292 (162V); IVS 2(−18insTT); rs1061170 (Y402H); and rs2274700 (A473A). In one embodiment,the sites are selected from: rs9427661 (−249T>C); rs9427662 (−20T>C);and rs12097550 (P46S). In a preferred embodiment, a diagnostic/screeningassay of the invention identifies the allele at least two polymorphicsites in the Factor H or CFHR5 gene. In a preferred embodiment, adiagnostic/screening assay of the invention identifies the allele atleast three polymorphic sites in the Factor H or CFHR5 gene. In apreferred embodiment, a diagnostic/screening assay of the inventionidentifies the allele at least four polymorphic sites in the Factor H orCFHR5 gene.

In some cases, the kit includes primers or probes (“oligonucleotides”)to identify the allele at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of thepolymorphisms in the Factor H or CFHR5 gene listed in TABLES 1A, 1B, 1C,11, 14 and 15. In one embodiment the kit includes primers or probes todetermine the allele at least one of the following polymorphic sites:rs529825; rs800292; rs3766404; rs1061147; rs1061170; rs203674; andoptionally including exon 22 (R120C). In one embodiment the kit includesprimers or probes to determine the allele at least one of the followingpolymorphic sites: rs529825; rs800292; intron 2 (IVS2 or insTT);rs3766404; rs1061147; rs1061170; exon 10A; rs203674; rs375046; andoptionally including exon 22 (R120C). In one embodiment the kit includesprimers or probes to determine the allele at least one of the followingpolymorphic sites: rs3753394; rs529825; rs800292; intron 2 (IVS2 orinsTT); rs3766404; rs1061147; rs1061170; rs2274700; rs203674; rs3753396;rs1065489; and optionally including exon 22 (R120C). In one embodiment,the sites are selected from: rs800292 (162V); IVS 2 (−18insTT);rs1061170 (Y402H); and rs2274700 (A473A). In one embodiment, the sitesare selected from: rs9427661 (−249T>C); rs9427662 (−20T>C); andrs12097550 (P46S).

The kit can include primers or probes to determine the allele at two ofthe above sites, at least three, at least four, at least five or atleast six. In one embodiment the primers or probes distinguish allelesat rs529825. In one embodiment the primers or probes distinguish allelesat rs800292. In one embodiment the primers or probes distinguish allelesat rs3766404. In one embodiment the primers or probes distinguishalleles at rs1061147. In one embodiment the primers or probesdistinguish alleles at rs1061170. In one embodiment the primers orprobes distinguish alleles at rs203674. In one embodiment; the primersor probes distinguish alleles at exon 22 (R1210C). In one embodiment theprimers or probes distinguish alleles at rs529825 and rs800292. In oneembodiment the primers or probes distinguish alleles at two or three ofrs1061147, rs1061170 and rs203674. In one embodiment the primers orprobes distinguish alleles at least one of rs529825 and rs800292; andrs3766404; and at least one of rs1061147, rs1061170 and rs203674. In oneembodiment the primers or probes distinguish alleles at rs529825,rs800292, rs3766404, rs1061170 and rs203674. In one embodiment, theprimers or probes distinguish alleles at exon 22 (R1210C) and at: (a)rs529825; rs800292; rs3766404; rs1061147; rs1061170; rs203674; rs529825rs800292; (b) at two or three of rs1061147, rs1061170 and rs203674; atrs529825 and rs800292, rs3766404, and two or three of rs1061147,rs1061170 and rs203674; or at rs529825, rs800292, rs3766404, rs1061170and rs203674. In one embodiment the primers or probes distinguishalleles at least one of rs529825 and rs800292; and rs3766404; and atleast one of rs1061147, rs1061170 and rs203674. In one embodiment theprimers or probes distinguish alleles at rs529825, rs800292, rs3766404,rs1061170 and rs203674.

The kit can include primers or probes to determine the allele at two ofthe above sites, or at least three of the above sites. In oneembodiment, the primers or probes distinguish alleles at rs800292. Inone embodiment, the primers or probes distinguish alleles at rs1061170.In one embodiment, the primers or probes distinguish alleles at exon 22(R1210C). In one embodiment, the primers or probes distinguish allelesat exon 22 (R1210C) and rs800292 and/or exon 22 and rs1061170 and exon22. In one embodiment, the primers or probes distinguish alleles atrs800292, rs1061170 and exon 22 (R1210C).

The kit can include primers or probes to determine the allele at two ofthe above sites, at least three, at least four, at least five or atleast six. In one embodiment the primers or probes distinguish allelesat rs529825. In one embodiment the primers or probes distinguish allelesat rs800292. In one embodiment the primers or probes distinguish allelesat intron 2 (IVS2 or insTT). In one embodiment the primers or probesdistinguish alleles at rs3766404. In one embodiment the primers orprobes distinguish alleles at rs1061147. In one embodiment the primersor probes distinguish alleles at rs1061170. In one embodiment theprimers or probes distinguish alleles at rs2274700. In one embodimentthe primers or probes distinguish alleles at exon 10A. In one embodimentthe primers or probes distinguish alleles at rs203674. In one embodimentthe primers or probes distinguish alleles at rs375046. In one embodimentthe primers or probes distinguish alleles at exon 22 (R1210C). In oneembodiment the primers or probes distinguish alleles at rs529825 andrs800292. In one embodiment the primers or probes distinguish alleles attwo or three of rs1061147, rs1061170 and rs203674. In one embodiment theprimers or probes distinguish alleles at rs529825 and rs800292, atintron 2, at rs3766404, at two or three of rs1061147, rs1061170 andrs203674, at rs2274700, at exon OA, and at rs375046. In one embodimentthe primers or probes distinguish alleles at rs529825, rs800292, intron2 (IVS2 or insTT), rs3766404, rs1061170, rs2274700, exon OA, rs203674,and rs375046. In one embodiment, the primers or probes distinguishalleles at exon 22 (R1210C) and at any one or more of rs529825;rs800292; intron 2 (IVS2 or insTT); rs3766404; rs1061147; rs1061170;rs2274700, exon 10A; rs203674; rs375046; rs529825 and rs800292. In oneembodiment, the primers or probes distinguish alleles at exon 22(R1210C) and at: (a) any two or three of rs1061147, rs1061170 andrs203674; (b) at rs529825 and rs800292, intron 2 (IVS2 or insTT),rs3766404, two or three of rs1061147, rs1061170 and rs203674, rs2274700,exon 10A, and rs375046; or at rs529825, rs800292, intron 2 (IVS2 orinsTT), rs3766404, rs1061170, rs2274700, exon 10A, rs203674, andrs375046.

In one embodiment the kit contains probes or primers for detecting atleast one variation in the Factor H gene as well as probes or primersfor detecting at least one variation in the CHFR-5 gene. In thisembodiment, the kit optionally contains probes or primers for detectingmore than on variation in either or both of the Factor H and CHFR-5genes, such as two, three or more than three variations.

A number of assay formats are known for determining haplotypes and canbe adapted to the present invention. See, e.g., Görgens et al., 2004,One-Step Analysis of Ten Functional Haplotype Combinations of the BasicRET Promoter with a LightCycler Assay” Clinical Chemistry 50:1693-1695;Dawson, 1989, “Carrier identification of cystic fibrosis by recombinantDNA techniques.” Mayo Clin Proc 64:325-34; Lee et al., 2005, “Gene SNPsand mutations in clinical genetic testing: haplotype-based testing andanalysis.” Mutat Res. 573:195-204.

In one embodiment, the primers or probes distinguish alleles at (a) anyone or more of rs529825; rs800292; rs3766404; rs1061147; rs1061170; andrs203674; (b) any one of more of intron 2 (IVS2 or insTT); rs2274700;exon 10A; and rs375046; (c) one or both of rs529825 and rs800292; (d)one or more of rs1061147, rs1061170 and rs203674; (e) at least one ofrs529825 and rs800292; and rs3766404; and at least one of rs1061147,rs1061170 and rs203674; (f) at least rs529825, rs800292, rs3766404,rs1061170, and rs203674; (g) exon 22 (R1210C); (h) exon 22 (R1210C) andany of (a)-(g); or (i) any one or more of rs529825; rs800292; rs3766404;rs1061147; rs1061170; rs203674; intron 2 (IVS2 or insTT); rs2274700;exon 10A; rs375046; and exon 22 (R1210C) and any one or more ofrs9427661, rs9427662 and rs12097550. In one embodiment, the kits includeoligonucleotide sufficient to distinguish any combination of alleles atthe sites listed below in the context of devices.

The kits may include antibodies that specifically recognize the Factor Hor CFHR5 polypeptide. The Factor H- or CFHR5-specific antibodies mayrecognize the normal, functional Factor H or CFHR5 polypeptide orvariant Factor H or CFHR5 polypeptides in which one or morenon-synonymous single nucleotide polymorphisms (SNPs) are present in theFactor H or CFHR5 coding region.

XII. Diagnostic Devices

Devices and reagents useful for diagnostic, prognostic, drug screening,and other methods are provided. In one aspect, a device comprisingimmobilized primer(s) or probe(s) specific for one or more Factor Hand/or CFHR5 polymorphic sites is provided. In an embodiment the FactorH and/or CFHR5 polymorphic sites are those described herein asassociated with AMD.

In one aspect, a device comprising immobilized primer(s) or probe(s)specific for one or more Factor H and/or CFHR5 gene products(polynucleotides or proteins) is provided. The primers or probes canbind polynucleotides (e.g., based on hybridization to specificpolymorphic sites) or polypeptides (e.g., based on specific binding to avariant polypeptide).

In one embodiment, an array format is used in which a plurality (atleast 2, usually at least 3 or more) of different primers or probes areimmobilized. The term “array” is used in its usual sense and means thateach of a plurality of primers or probes, usually immobilized on asubstrate, has a defined location (address) e.g., on the substrate. Thenumber of primers or probes on the array can vary depending on thenature and use of the device. For example, a dipstick format array canhave as few as 2 distinct primers or probes, although usually more than2 primers or probes, and often many more, will be present. One methodfor attaching the nucleic acids to a surface is by making high-densityoligonucleotide arrays (see, Fodor et al., 1991, Science 251:767-73;Lockhart et al., 1996, Nature Biotech 14:1675; and U.S. Pat. Nos.5,578,832; 5,556,752; and 5,510,270). It is also contemplated that, insome embodiments, a device comprising a single immobilized probe can beused.

In one embodiment, an array format is used in which a plurality (atleast 2, usually at least 3 or more) of different primers or probes areimmobilized. The term “array” is used in its usual sense and means thateach of a plurality of primers or probes, usually immobilized on asubstrate, has a defined location (address) e.g., on the substrate. Thenumber of primers or probes on the array can vary depending on thenature and use of the device.

In one embodiment, the immobilized probe is an antibody or other FactorH or CFHR5 binding moiety.

It will be apparent to the skilled practitioner guided by thisdisclosure than various polymorphisms and haplotypes can be detected toassess the propensity of an individual to develop a Factor H relatedcondition. The following examples and combinations are provided forillustration and not limitation. In some cases, the array includesprimers or probes to identify the allele at 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or all of the polymorphisms in the Factor H or CFHR5 gene listed inTABLES 1A, 1B, 1C, 11, 14 and 15. In one embodiment the array includesprimers or probes to determine the allele at least one of the followingpolymorphic sites: rs529825; rs800292; rs3766404; rs1061147; rs1061170;rs203674; and optionally including exon 22 (R1210C). In one embodimentthe array includes primers or probes to determine the allele at leastone of the following polymorphic sites: rs529825; rs800292; intron 2(IVS2 or insTT); rs3766404; rs1061147; rs1061170; exon 10A; rs203674;rs375046; and optionally including exon 22 (R1210C). In an embodimentthe array includes primers or probes to determine the allele at leastone of the following polymorphic sites: (a) rs3753394; (b) rs529825; (c)rs800292; (d) intron 2 (IVS2 or insTT); (e) rs3766404; (f) rs1061147;(g) rs1061170; (h) rs2274700; (i) rs203674; (j) rs3753396; (k)rs1065489; and optionally including exon 22 (R1210C). In one embodiment,the array includes primers or probes to determine the allele at leastone of the following polymorphic sites: rs800292 (162V); IVS 2(−18insTT); rs1061170 (Y402H); and rs2274700 (A473A). In one embodiment,the array includes primers or probes to determine the allele at leastone of the following polymorphic sites: rs9427661 (−249T>C); rs9427662(−20T>C); and rs12097550 (P46S).

The array can include primers or probes to determine the allele at twoof the above sites, at least three, at least four, at least five or atleast six. In one embodiment the primers or probes distinguish allelesat rs529825. In one embodiment the primers or probes distinguish allelesat rs800292. In one embodiment the primers or probes distinguish allelesat rs3766404. In one embodiment the primers or probes distinguishalleles at rs1061147. In one embodiment the primers or probesdistinguish alleles at rs1061170. In one embodiment the primers orprobes distinguish alleles at rs203674. In one embodiment the primers orprobes distinguish alleles at exon 22 (R1210C). In one embodiment theprimers or probes distinguish alleles at rs529825 and rs800292. In oneembodiment the primers or probes distinguish alleles at two or three ofrs1061147, rs1061170 and rs203674. In one embodiment the primers orprobes distinguish alleles at rs529825 and rs800292, at rs3766404, twoor three of rs1061147, rs1061170 and rs203674. In one embodiment theprimers or probes distinguish alleles at rs529825, rs800292, rs3766404,rs1061170 and rs203674. In one embodiment, the primers or probesdistinguish alleles at exon 22 (R1210C) and at rs529825; at rs800292; atrs3766404; at rs1061147; at rs1061170; at rs203674; at rs529825 andrs800292; at two or three of rs1061147, rs10611170 and rs203674; atrs529825 and rs800292, rs3766404, and two or three of rs1061147,rs1061170 and rs203674; or at rs529825, rs800292, rs3766404, rs1061170and rs203674. In one embodiment, the primers or probes distinguishalleles at (a) any one or more of rs529825; rs800292; rs3766404;rs1061147; rs1061170; and rs203674; (b) any one of more of intron 2(IVS2 or insTT); rs2274700; exon 10A; and rs375046; (c) one or both ofrs529825 and rs800292; (d) one or more of rs1061147, rs1061170 andrs203674; (e) at least one of rs529825 and rs800292; and rs3766404; andat least one of rs1061147, rs1061170 and rs203674; (f) at leastrs529825, rs800292, rs3766404, rs1061170, and rs203674; (g) exon 22(R1210C); (h) exon 22 (R1210C) and any of (a)-(g); or (i) any one ormore of rs529825; rs800292; rs3766404; rs1061147; rs1061170; rs203674;intron 2 (IVS2 or insTT); rs2274700; exon 10A; rs375046; and exon 22(R1210C.) and any one or more of rs9427661, rs9427662 and rs 12097550.

The array can include primers or probes to determine the allele at twoof the above sites, at least three, at least four, at least five or atleast six. In one embodiment the primers or probes distinguish allelesat rs529825. In one embodiment the primers or probes distinguish allelesat rs800292. In one embodiment the primers or probes distinguish allelesat intron 2 (IVS2 or insTT). In one embodiment the primers or probesdistinguish alleles at rs3766404. In one embodiment the primers orprobes distinguish alleles at rs1061147. In one embodiment the primersor probes distinguish alleles at rs1061170. In one embodiment theprimers or probes distinguish alleles at exon 10A. In one embodiment theprimers or probes distinguish alleles at rs2274700. In one embodimentthe primers or probes distinguish alleles at rs203674. In one embodimentthe primers or probes distinguish alleles at rs375046. In one embodimentthe primers or probes distinguish alleles at exon 22 (R1210C). In oneembodiment the primers or probes distinguish alleles at rs529825 andrs800292. In one embodiment the primers or probes distinguish alleles attwo or three of rs1061147, rs1061170 and rs203674. In one embodiment theprimers or probes distinguish alleles at of rs529825 and rs800292, atintron 2, at rs3766404, at two or three of rs1061147, rs1061170 andrs203674, at exon 10A, at rs2274700, and at rs375046. In one embodimentthe primers or probes distinguish alleles at rs529825, rs800292, intron2 (IVS2 or insTT), rs3766404, rs1061170, exon 10A, rs2274700, rs203674,and rs375046. In one embodiment, the primers or probes distinguishalleles at exon 22 (R1210C) and at either at rs529825; at rs800292; atintron 2 (IVS2 or insTT); at rs3766404; at rs1061147; at rs1061170; atrs2274700, at exon 10A; at rs203674; at rs375046; at rs529825 and rs800292; at two or three of rs1061147, rs1061170 and rs203674; atrs529825 and rs800292, intron 2 (IVS2 or insTT), rs3766404, two or threeof rs1061147, rs1061170 and rs203674, rs2274700, exon 10A, and rs375046;or at rs529825, rs800292, intron 2 (IVS2 or insTT), rs3766404,rs1061170, rs2274700, exon 10A, rs203674, and rs375046. In oneembodiment, the device distinguishes any combination of alleles at thesites listed above in the context of kits.

In one embodiment, the substrate comprises fewer than about 1000distinct primers or probes, often fewer than about 100 distinct primersor probes, fewer than about 50 distinct primers or probes, or fewer thanabout 10 distinct primers or probes. As used in this context, a primeris “distinct” from a second primer if the two primers do notspecifically bind the same polynucleotide (i.e., such as cDNA primersfor different genes). As used in this context, a probe is “distinct”from a second probe if the two probes do not specifically bind the samepolypeptide or polynucleotide (i.e., such as cDNA probes for differentgenes). Primers or probes may also be described as distinct if theyrecognize different alleles of the same gene (i.e., CFH or CFHR5). Thus,in one embodiment diagnostic devices of the invention detect alleles ofCFH only, CFHR5 only, CFH and CFHR5 only, or CFH, CFHR5 and up to 20,preferably up to 10, or preferably up to 5 genes other than CFH and/orCFHR5. That is, the device is particularly suited to screening for AMDand related complement-associated diseases. In one embodiment, thedevice comprises primers or probes that recognize CFH and/or one or moreof CFHR1-5 only. In a related embodiment, the device contains primersand probes for up to 20, preferably up to 10, or preferably up to 5other genes than CFH or CFHR1-5.

In one embodiment, the immobilized primer(s) is/are an allele-specificprimer(s) that can distinguish between alleles at a polymorphic site inthe Factor H or CHRF5 gene. Exemplary allele-specific primers toidentify alleles at polymorphic sites in the Factor H gene are shown inTABLE 16A. The immobilized allele-specific primers hybridizepreferentially to nucleic acids, either RNA or DNA, that have sequencescomplementary to the primers. The hybridization may be detected byvarious methods, including single-base extension with fluorescencedetection, the oligonucleotide ligation assay, and the like (see Shi, M.M., 2001, Enabling large-scale pharmacogenetic studies byhigh-throughput mutation detection and genotyping technologies” Clin.Chem. 47(2): 164-172). Microarray-based devices to detect polymorphicsites are commercially available, including Affymetrix (Santa Calar,Calif.), Protogene (Menlo Park, Calif.), Genometrix (The Woodland,Tex.), Motorola BioChip Systems (Northbrook, Ill.), and PerlegenSciences (Mountain View, Calif.).

In one embodiment, the immobilized probe(s) is/are an allele-specificprobe(s) that can distinguish between alleles at a polymorphic site inthe Factor H or CFHR5 gene. Exemplary allele specific probes to identifyalleles at polymorphic sites in the Factor H gene are shown in TABLE16B. The immobilized allele-specific probes hybridize preferentially tonucleic acids, either RNA or DNA, that have sequences complementary tothe probes. The hybridization may be detected by various methods,including fluorescence of hybridized nucleic acids (see Shi, M. M.,2001, Enabling large-scale pharmacogenetic studies by high-throughputmutation detection and genotyping technologies. Clin. Cliem.47(2):164-172). Microarray-based devices to detect polymorphic sites arecommercially available, including Affymetrix (Santa Calar, Calif.),Protogene (Menlo Park, Calif.), Genometrix (The Woodland, Tex.),Motorola BioChip Systems (Northbrook, Ill.), and Perlegen Sciences(Mountain View, Calif.).

In certain embodiments probes or primers specific for particular SNPsand variations are excluded from a kit or a device of the invention. Forexample, in some embodiments, a kit or device does not include one ormore of the following SNPs can be excluded: (i) rs529825; (ii) rs900292;(iii) intron 2 (IVS2 or insTT); (iv) rs3766404; (v) rs1061147; (vi)rs1061170; (vii) rs2274700; (viii) exon 10A; (ix) rs203674; (x)rs375046; (xi) rs3753396; (xii) rs1065489; or (xiii) exon 22 (R1210C).

XIII. Examples Example 1 Common Haplotype in the Factor H Gene (HF1/CFH)Predisposes Individuals to Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is the most frequent cause ofirreversible blindness in the elderly in developed countries, affectingmore than 50 million individuals worldwide. Our previous studiesimplicated activation of the alternative complement pathway in theformation of ocular drusen, the hallmark lesion of AMD. We have alsoshown that macular drusen in AMD patients are indistinguishable fromthose that form at an early age in individuals withmembranoproliferative glomerulonephritis type 2 (MPGNII), a diseasecharacterized by uncontrolled activation of the alternative pathway ofthe complement cascade. Here we show that Factor H protein (HF1), themajor inhibitor of the alternative complement pathway, accumulateswithin drusen, and is synthesized locally by the retinal pigmentepithelium. Previous linkage analyses identified chromosome 1q25-32,which harbors the Factor H gene (HF1/CFH), as a major AMD susceptibilitylocus. We analyzed HF1 for genetic variation in two independent cohortscomprised of approximately 900 AMD cases and 400 matched controls. Wefind a highly significant association of 8 common HF1 SNPs with AMD inthese cohorts; two common missense variants exhibit highly significantassociations (I62V; χ²=36.1, p=3.2×10⁻⁷ and Y402H; χ²=54.4,p=1.6×10⁻¹³). Haplotype analysis suggests that multiple HF1 variantsconfer either an elevated or a reduced risk of AMD. One common at-riskhaplotype is present at a frequency of 49% in AMD cases and 26% incontrols (OR=2.67, 95% CI [1.80-2.85]). Homozygotes for this haplotypeaccount for 22.1% of cases and 5.1% of controls (OR=5.26, 95% CI[2.84-9.76]). Several protective haplotypes are also identified(OR=0.44-0.55). Further strengthening these data is the finding of therisk haplotype in 70% of MPGNII patients. We propose that geneticallypre-determined variation in regulators of the complement system, whencombined with triggering events such as infection, underlie a majorproportion of AMD in the human population.

Introduction

Age-related macular degeneration (AMD) is the leading cause ofirreversible vision loss in the developed world (Klein et al., 2004; vanLeeuwen et al., 2003), affecting 15% of individuals over the age of 60or an estimated 600 million individuals. AMD is characterized by aprogressive loss of central vision attributable to degenerative andneovascular changes which occur at the interface between the neuralretina and the underlying choroid. At this location lie thephotoreceptors, the adjacent retinal pigmented epithelium (RPE), abasement membrane complex known as Bruch's membrane BM), and a networkof choroidal capillaries.

The prevailing view is that AMD is a complex disorder attributable tothe interaction of multiple genetic and environmental risk factors(Klein et al., 2003; Tuo et al., 2004). Familial aggregation studiesindicate that a genetic component can be identified in up to 25% of thecases (Klaver et al., 1998). As such, AMD appears to be a product of theinteraction between multiple susceptibility loci rather than acollection of single-gene disorders. The number of loci involved, theattributable risk conferred, and the interactions between various lociremain obscure.

Linkage analyses and candidate gene screening have provided limitedinsight into the genetics of AMD. Reliable associations of one gene withincreased risk, ABCA4 (Allikmets et al., 1997; Allikmets, 2000), and onegene with decreased risk, ApoE4 (Klaver et al., 1998; Souied et al.,1998), have been reported. Several groups have reported results fromgenome-wide linkage analyses (Tuo et al., 2004; Weeks et al., 2001). Todate, linkage of one AMD phenotype (ARMD1; MIM603075) to a specificchromosomal region, 1q25-q31, has been documented (Klein et al., 1998).HEMICENTIN-1, also known as Fib16, has been tentatively identified asthe causal gene (Schultz et al., 2003), although it does not account fora significant disease load (Abecasis et al., 2004; Hayashi et al.,2004). The identification of overlapping loci on chromosome 1q byseveral groups (Weeks et al., 2001; Iyengar et al., 2003) indicates thatthis locus is likely to harbor a major AMD-associated gene.

In AMD, as well as many other diseases such as Alzheimer's disease(Akiyama et al., 2000), atherosclerosis (Torzewski et al., 1997), andglomerulonephritis (Schwertz et al., 2001), characteristic lesions anddeposits contribute to the pathogenesis and progression of the disease.Although the molecular pathogenesis of these diseases may be diverse,the deposits contain many shared molecular constituents that areattributable, in part, to local inflammation and activation of thecomplement cascade, a key element of host defense in the innate immunesystem. Drusen are the hallmark deposits associated with early AMD, andrecent studies have implicated local inflammation and activation of thecomplement cascade in their formation as well (Hageman et al., 1999;Espinosa-Heidmann et al., 2003). Drusen contain a variety of complementactivators, inhibitors, activation-specific complement fragments, andterminal pathway components including the membrane attack complex (MAC),the lytic complex formed as a consequence of complement activation. TheMAC is potentially lethal to host cells and tissues as well as foreignpathogens.

Many individuals with membranoproliferative glomerulonephritis type II(MPGNII), a rare kidney disease characterized by uncontrolled systemicactivation of the alternative activation pathway of complement, alsodevelop ocular drusen in the macula that are indistinguishable incomposition and appearance from those in AMD (Mullins et al., 2001;O'brien et al., 1993; McAvoy et al., 2004). Furthermore, one patientdiagnosed with MPGNII harbors a mutation in HF1 (HF1), a major inhibitorof the alternative pathway of complement activation (Zipfel, personalcommunication). Additionally, individuals in a few extended familieswith MPGNIII, a related disorder, show linkage to a region of chromosomemapped in 1q31-32 (Neary et al., 2002) that overlaps the locusidentified in genome-wide linkage studies for AMD. Collectively, thesefindings provided the impetus for examining whether HF1 is involved inthe development of AMD and MPGNII.

In this investigation, we determined the frequencies of HF1 sequencevariants in AMD and MPGNII patients and matched controls, and analyzedtheir association with disease phenotype. We also examined HF1transcription and the distribution of HF1 protein in the macularRPE-choroid complex from normal and AMD donors.

Methods

Patients, Phenotyping and DNA—Two independent groups of AMD cases andage-matched controls were used for this study. All participatingindividuals were of European-American descent, over the age of 60, andenrolled under IRB approved protocols following informed consent. Thesegroups were comprised of 404 unrelated patients with clinicallydocumented AMD (mean age 79.5±7.8) and 131 unrelated, controlindividuals (mean age 78.4±7.4; matched by age and ethnicity) from theUniversity of Iowa, and 550 unrelated patients with clinicallydocumented AMD (mean age 71.32±8.9 years), and 275 unrelated, matched byage and ethnicity, controls (mean age 68.84±8.6 years) from the ColumbiaUniversity. Patients were examined by indirect ophthalmoscopy andslit-lamp microscopy by retina fellowship-trained ophthalmologists.

Dr. Caroline Klaver, and later individuals trained by Dr. Klaver, gradedfundus photographs at both institutions according to a standardized,international classification system (Bird et al., 1995). Controlpatients were selected and included if they did not exhibit anydistinguishing signs of macular disease or have a known family historyof AMD. The AMD patients were subdivided into phenotypiccategories—early AMD (eAMD), geographic atrophy (GA) and exudative (CNV)AMD—based on the classification of their most severe eye at the time oftheir entry into the study. The University of Iowa eAMD and GA caseswere further subdivided into distinct phenotypes (RPE changes alone, >10macular hard drusen, macular soft drusen, BB (cuticular) drusen, PED,“Cherokee” atrophy, peninsular geographic atrophy and pattern geographicatrophy). The earliest documented phenotype for all cases was alsorecorded and employed in the analyses.

Genomic DNA was generated from peripheral blood leukocytes collectedfrom case and control subjects using QIAamp DNA Blood Maxi kits (Qiagen,Valencia, Calif.).

Rapanui—Following an informed consent process approved by the Unidad deBioetica, Ministerio de Salud (Santiago, Chile), 447 (66% female; 34%male) Easter Island inhabitants were provided a complete eye examinationthat included a dilated funduscopic examination. Medical, family andophthalmic histories were taken and records and assistance from localphysicians and community leaders were used to classify the ethnicity ofthe subjects. 49% of those patients examined were pure Rapanui, 9% wereadmixed (mixture of Rapanui and European, Chilean, Mapuchi and/or recentPolynesian), and 42% were continental (largely Chilean European).Peripheral venous blood and sera were collected from 201 of the olderindividuals; 114 of these individuals were pure Rapanui (108 were >50years old; 89 were >60 years old). DNA from 60 of the pure Rapanuiinhabitants and 13 of the Chilean residents over the age of 65 was usedin this study.

Human Donor Eyes—Human donor eyes were obtained from the Iowa Lions EyeBank (Iowa City, Iowa), the Oregon Lions Eye Bank (Portland, Oreg.) andthe Central Florida Lions Eye and Tissue Bank (Tampa, Fla.) within fivehours of death. Gross pathologic features of these eyes, as well asfundus photographs and angiograms, when available, were read andclassified by retinal specialists. Fundi were graded according to amodified version of the International AMD grading system (Bird et al.,1995) by at least two individuals.

Total RNA was prepared from retina, RPE/choroid, and RPE cells derivedfrom eyes using an RNeasy Mini Kit (Qiagen, Valencia, Calif.). GenomicDNA was sheared using a QiaShredder (Qiagen, Valencia, Calif.) andresidual genomic DNA digested with DNAse (Promega). RNA integrity wasassessed using an Agilent BioAnalyzer.

DNA derived from 38 unrelated donors with clinically documented AMD(mean age 81.5±8.6) and 19 unrelated, control donors (mean age 80.5±8.8;matched by age and ethnicity) were employed for SSCP analyses and toassess potential genotype-phenotype correlations.

Immunohistochemistry—Wedges of posterior poles, including the oraserrata and macula were fixed and processed as described previously(Hageman et al., 1999). Some posterior poles were embedded directly inOCT without prior fixation. Tissues were sectioned to a thickness of 6-8μm on a cryostat and immunolabeling was performed as describedpreviously (Hageman et al., 1999. Adjacent sections were incubated withsecondary antibody alone, to serve as controls. Some immunolabeledspecimens were prepared and viewed by confocal laser scanningmicroscopy, as described previously (Anderson et al., 2002).

Polymerase Chain Reaction (PCR)—First strand cDNA was synthesized fromtotal RNA using Superscript reverse transcriptase (Gibco BRL) and randomhexamers. PCR reactions were carried out using the following primersets: FH1 (exon 8 to exon 10) 5′-GAACATTTTGAGACTCCGTC-3′ [SEQ ID NO:324]and 5′-ACCATCCATCTTTCCCAC-3′ [SEQ ID NO:325]; FH1 (exon 9 to exon 10)5′-TCCTGGCTACGCTCTTC-3′ [SEQ ID NO:326] and 5′-ACCATCCATCTTTCCCAC-3′[SEQ ID NO:325]; HFL1 (exon 8 to exon 10) 5′-TCCGTCAGGAAGTTACTGG-3′ [SEQID NO:327] and 5′-AGTCACCATACTCAGGACCC-3′ [SEQ ID NO:328]; HFL1 (exon 9to exon 10), 5′-GGCTACGCTCTTCCAAAAG-3′ [SEQ ID NO:329] and5′-AGTCACCATACTCAGGACCC-3′ [SEQ ID NO:330]. PCR primers (IDT,Coralville, Iowa) were designed using MacVector software (San Diego,Calif.). Reaction parameters were one cycle at 94° C. for 3 minutes, 40cycles at 94° C. for 45 sec, 51.4° C. (FH1)/55° C. (HFL1) for 1 min, 72°C. for 1 min, and one cycle at 72° C. for 3 min. The PCR products wererun on 2% agarose gels and recorded using a Gel Doc 2000™ DocumentationSystem accompanied by Quantity One® software (Bio-Rad, Hercules,Calif.).

Microarray Analyses: DNA microarray analyses were performed using totalRNA extracted from native human RPE or the RPE-Choroid complex (RNeasyminikit, Qiagen, Valencia, Calif.) collected within <5 hours of death.Three different platforms were used: an 18,380 non-redundant DNAmicroarray (Incyte Pharmaceuticals; St. Louis, Mo.); the Affymetrix genechip system; and a Whole Human Genome or Human 1A V2 oligo-microarray(Agilent Inc., Palo Alto, Calif.). The individual protocols followedeach of the manufacturer's instructions. For the Incyte analyses, cDNAderived from 6 mm punches of macular and mid-peripheral regions waslabeled with 33-P in a random-primed reaction, purified and hybridizedto the Nylon-based arrays containing 18,380 non-redundant cDNAs. Themembranes were phosphoimaged, signals were normalized and data wereanalyzed using the Genome Discovery Software package. For the Affymetrixanalyses, RPE and RPE/choroid (from 6-8 mm macular and peripheralpunches) cRNA was hybridized directly to Affymetrix GeneChips (HG-U133A)using standardized protocols. These procedures were conducted in theUniversity of Iowa DNA core facility, which is equipped with a fluidicsstation and a GeneArray scanner. The Agilent data was obtained frompunches of the macula and mid-periphery. CY3 and CY5 labeled amplifiedcRNA derived from macula and peripheral RPE/choroid was generated usingan Agilent Low Input RNA Amplification Kit using the macular andperipheral RNA from the same donor. The Agilent array data werecollected from 3 normal young donors, 3 AMD donors, and 3 age-matchednon-AMD controls using a VersArray Scanner; data were quantified usingthe VersArray Analyzer Software (BioRad). The median net intensity ofeach spot was calculated using global background subtraction and thedata was normalized using the local regression method.

Mutation Screening and Analysis—Coding and adjacent intronic regions ofHF1 (including exon 10A that is transcribed to generate the truncatedFHL1 isoform) were examined for variants using single-strandconformation polymorphism (SSCP) analyses, denaturing high performanceliquid chromatography (DHPLC) and direct sequencing. The remaining SNPswere typed by the 5′ nuclease (Taqman, ABI) methodology. Taqmangenotyping and association analyses were performed as described (Gold etal., 2004). Primers for SSCP, DHPLC and DNA sequencing analyses (TABLE5) were designed to amplify each exon and its adjacent intronic regionsusing MacVector software (San Diego, Calif.). PCR-derived amplicons werescreened for sequence variation by SSCP and DHPLC, as describedpreviously (Allikmets et al., 1997; Hayashi et al., 2004). All changesdetected by SSCP and DHPLC were confirmed by bidirectional sequencingaccording to standard protocols. Statistical analyses were performedusing chi-square and Fisher's exact tests.

Results Factor H at the RPE-Choroid Interface

The distribution of Factor H protein in the RPE/choroid complex from themacula and extramacula was assessed in eyes obtained from six donorswith a history of early AMD and three donors of similar age without AMDor drusen (FIGS. 1A-1L). In donors with AMD, intense HF1immunoreactivity (IR) is present in drusen, beneath the RPE (i.e. thesub-RPE space), and around the choroidal capillaries (FIGS. 1A-1D, 1E,1G). In the absence of the primary antibody, labeling in the RPE/choroidis absent (FIG. 1F). All of the Factor H antibodies labeled drusen tosome degree, in a homogeneous fashion (FIGS. 1C and 1E). One antibodyalso labeled substructural elements within drusen (FIGS. 1A and 1B).Such structures are also labeled using antibodies to the activatedcomplement component C3b/iC3b that binds HF1 (Anderson et al., 2004;Johnson et al., 2001). Factor H immunoreactivity is more robust indonors with AMD compared to age-matched controls; it is also morepronounced in the maculas of AMD donors than in the periphery (FIGS. 1Gand 1H). The anti-HF1 pattern in the macula (FIG. 1G) is highly similarto the anti-C5b-9 pattern (FIGS. 1I and 1K); in both cases, labelingtypically includes the choroidal capillaries. Extramacular locationsshow much less anti-C5b-9 immunoreactivity (FIG. 1J). Little or no C5b-9immunoreactivity in the RPE-choroid is observed in donors under the ageof 50 and without AMD (FIG. 1L).

Description of FIG. 1

(A-B) High magnification confocal immunofluorescence images from an 84year old male donor diagnosed with atrophic AMD. Anti-HF1 (AdvancedResearch Technologies) labeling of substructural elements (white arrows)in drusen and the sub-RPE space is imaged on the Cy2/fluoresceinchannel. The sub-RPE space is the extracellular compartment between thebasal RPE surface and the inner collagenous layer of Bruch's membrane.Such elements also display immunoreactivity (IR) using monoclonalantibodies directed against C3 fragments (iC3b, C3d, C3dg) that bindcovalently to complement activating surfaces (Johnson et al, 2001;2003). The intense anti-Factor H labeling in the lumens of choroidalcapillaries (asterisks) from this donor most likely reflects the highcirculating levels of HF1 in the bloodstream. Autofluorescent lipofuscingranules in the RPE cytoplasm are labeled on the Cy3/Texas Red channel.Magnification bars. A) 5 μm; B) 3 μm.

(C-D) Confocal immunofluorescence localization of HF1 in drusen and thesub-RPE space in an 83 year old male with AMD using a different HF1polyclonal antibody (Quidel) (Cy2/fluorescein channel; green). C) Inthis donor eye, the drusen (Dr) labeling pattern is homogeneous. D) Lowmagnification image of the RPE-choroid. Anti-HF1 IR is presentthroughout the choroid and in the sub-RPE space (arrows), the anatomicalcompartment where drusen and other deposits associated with aging andAMD form. Lipofuscin autofluorescence (Cy3 channel; red). Magnificationbars. C) 10 μm; D) 20 μm.

(E-F) Immunohistochemical localization of HF1 in drusen. E) Anti-HF1monoclonal antibody (Quidel) labeling, signified by the purple alkalinephosphatase reaction product, is apparent in drusen, along Bruch'smembrane, and on the choroidal capillary walls (arrows). F) Controlsection from the same eye. In the absence of the primary antibody, nolabeling is present. Brown pigmentation in the RPE cytoplasm and choroidis melanin. Magnification bars=10 μm.

(G-H) Immunolocalization of HF1 in the macula. G) Extensive labeling ispresent along BM, the choroidal capillary walls, and the intercapillarypillars (arrows) in a donor with AMD. H) Control section from the maculaof a donor without AMD; much less labeling is apparent in samestructures. Magnification bars=20 μm.

(I-J) Immunohistochemical localization of the complement membrane attackcomplex (C5b-9) in the RPE-choroid underlying the macula (FIG. 1I) andextramacula (FIG. 1J) from the same AMD donor eye. In the macula,intense anti-C5b-9 labeling is associated with drusen, Bruch's membrane,and the choroidal capillary endothelium. Anti-C5b-9 labeling outside themacula is restricted to a narrow zone in the vicinity of Bruchsmembrane. Brown pigment in the RPE cytoplasm and choroid representsmelanin pigmentation. Magnification bars=20 μm.

(K-L) Immunohistochemical location of C5b-9 in the macula from a donorwith AMD (FIG. 1K) and from a second donor without AMD (FIG. 1L). Brownpigmentation in the RPE cytoplasm and choroid represents melanin. Theanti-C5b-9 labeling is associated primarily with the choroidal capillarywalls (black arrows) and the intercapillary pillars (white arrows).Labeling is much more intense in the AMD eye. Note the strong similarityto the anti-HF1 labeling pattern in the macula from the same donor, asshown in Figure G. Magnification bars. K=15 μm; L=20 μm.

The Retinal Pigment Epithelium is a Local Source of Factor H

Expression of HF1 and FHL1 in the RPE, RPE/choroid and retina wasassessed by RT-PCR and DNA microarray analysis. Appropriately sized PCRproducts for both gene products are present in freshly isolated RPE andthe RPE/choroid complex, but not neural retina, in human eyes derivedfrom donors with and without AMD (FIG. 2). Primers were chosen withinexons 8, 9, 10 A (the exon employed to generate the truncated isoformFHL1) and 10 of the HF1 coding sequence. The PCR reactions wereperformed with cDNA prepared from RNA extracted from human neurosensoryretina (lanes 2), RPE and choroid (lanes 3), and freshly isolated RPEcells (lanes 4) derived from a donor with a clinically documentedhistory of AMD. Genomic DNA was employed as a template for amplification(lanes 5); no template was added to the mixtures depicted in lanes 6.Lanes 1 contains the 100 bp ladder. Amplimers spanning from exon 8 toexon 10 (left panel), and from exon 9 to exon 10 (right panel), of HF1and from exon 8 to exon 10A (left panel), and exon 8 to exon 10A (rightpanel), of FHL1 were of the expected sizes (376, 210, 424 and 248 bp,respectively). Transcripts for FHRs 1-5 are not detected in RPE orRPE/choroid, but FHRs 1-4 are detected in neural retina by RT-PCR (datanot shown).

Gene expression array data derived from three platforms confirm that HF1and FHL1 transcripts, but few if any of the HF1-related proteintranscripts (FHR1 being the possible exception), are expressed locallyby RPE and choroid cells. Data derived from Incyte arrays probed withRPE/choroid cDNA derived from nine donors with AMD and three age-matchedcontrols show elevated levels that average 2-3 times that of HF1 mRNA inthe donors with AMD. There is also a trend toward slightly higher levelsin the macula regions as compared to the extramacular regions, althoughthe difference is not statistically significant. The data generated fromthe examination of isolated RPE and adjacent RPE/choroid preparationsfrom two donors with AMD and two age-matched control donors usingAffymetrix arrays confirm the presence of HF1 transcripts in thesetissues and shows that a significant proportion of the HF1 message ispresent within the RPE layer (data not shown).

Variants in HF1 are Associated with AMD and MPGN II

To test whether allele variants of HF1 gene are associated with AMD, all22 coding exons and 50-100 bp flanking intronic sequences were screened,in a cohort of 404 AMD patients and 131 matched controls at theUniversity of Iowa. A total of 26 sequence variants are detected; 17SNPs in the coding region (cSNPs), including 5 synonymous and 12non-synonymous substitutions, and 9 intronic SNPs (some of the variantsshown in FIG. 3). FIG. 3 shows the approximate locations of 11 SNPs usedin the analysis, the 20 short consensus repeats (SCRs), and the linkagedisequilibrium (LD) blocks, and the approximate binding sites forpathogens and other substrates are depicted below the diagram based onpreviously published data (Zipfel et al., 2002; Rodriguez de Cordoba etal., 2004). cSNPs included previously described common non-synonymousvariants, such as I62V in exon 2, Y402H in exon 9, and D936E in exon 18(FIG. 3). An example of a common intronic SNP with a potentiallyfunctional effect is the IVS2-18insTT variant. Five rare (<0.5%)variants are also detected (data not shown) in both AMD patients andcontrols, excluding the possibility of the disease phenotype beingcaused by rare HF1 alleles (i.e., disease-causing mutations). Detailedgenotyping data were obtained for 6 SNPs in some or all of the 404patients and 131 controls (TABLES 4 and 6A-6C) and association analyseswere performed using a case-control study design. Highly significantassociations are found with several individual variants, including I62V(χ²=15.0, p=1.1×10⁻⁴) and Y402H (χ²=49.4, p=2.1×10⁻¹²). The strongestassociation with AMD in this cohort is observed with a synonymous A473Avariant in exon 10, resulting in an odds ratio (OR) of 3.42 (95%confidence interval (CI) [2.27-5.15]).

These results were confirmed in an independent cohort of AMD patients(n=550) and matched controls (n=275) obtained at Columbia University,New York (TABLE 4). The same two non-synonymous SNPs are also highlyassociated with AMD in this second cohort (I62V; χ²=36.1, p=3.2×10⁻⁷ andY402H; χ²=54.4, p=1.6×10⁻¹³). In addition, several other intronic SNPswere selected based on frequency and the availability of commercialassays (for a total of 11 SNPs). The strongest association in thiscohort is observed with SNP rs203674 in intron 10 (χ²=66.1,p=4.29×10⁻¹⁶). This variant shows an OR of 2.44 with AMD (95%CI=1.97-3.03). Although the OR is modest, the variant is very common;30.5% of the cases are homozygous for allele B, but only 12.9% of thecontrols. The Q672Q and D936E alleles in exon 13 and 18 show nostatistically significant association, suggesting that variation in theN-terminal half of HF1, which includes domains involved in pathogen andsubstrate molecule recognition (FIG. 3, also see below), are associatedwith AMD. The two sets of data are strikingly similar, in that thegenotyped SNPs are not only associated with AMD in a highly significantfashion, but the frequencies and extent of association are very similarin the two cohorts (TABLES 4 and 6).

The association is highly significant when the entire AMD patientcohorts are compared to controls (TABLE 4). When the majorsub-phenotypes of AMD, such as the early AMD (eAMD, characterized bymacular drusen and/or pigmentary abnormalities), CNV (neovascularmembranes and/or disciform scars), and GA (geographic atrophy) areanalyzed separately, the association is especially prominent in cases ofeAMD and CNV. The GA group shows some deviation from the general trendin some cases, especially with the haplotype defined by exon 13 (Q672Q)and 18 (D936E) alleles (data not shown). While this deviation may besignificant in terms of varying etiology, it did not reach statisticalsignificance, most likely due to relatively small numbers of patientswith GA.

Linkage disequilibrium (LD) analysis showed extensive LD across theentire HF1 gene (TABLE 2 and FIG. 3). Three SNPs in the exon 2-3 regionare in virtually complete LD as are the A307A and Y402H variants inexons 7 and 9, and the Q672Q and D936E variants in exon 13 and 18 (TABLE6 and FIG. 5). Haplotype estimation in cases and controls identified themost frequent at-risk haplotype in 49% of cases versus only in 26% ofcontrols (OR=2.93 95% CI [2.29-3.74]). Homozygotes for this haplotypeare present in 22.1% of the Columbia cases and 5.1% of the controls(OR=5.26, 95% CI [2.84-9.76]). Two common protective haplotypes arefound in 30% of controls and 18% of cases (OR=0.476 95% CI [0.349-0.650]and OR=0.472, 95% CI [0.320-0.698]). These haplotypes differ only in theexon 2-3 locus SNPs and the intron 10 SNP. As shown in FIG. 4 and TABLE2, these protective haplotypes are closely related to each other and areboth at least five steps away from the risk haplotype. Interestingly,the 3 SNPs, (promoter −257C>T, A473A, and D936E) previously shown to beassociated with HUS are all on one relatively common haplotype (12%)that is neutral for AMD risk (see the discussion below). For each SNP weidentified the base present in the consensus chimpanzee genome. Thehaplotype generated represents the likely ancestral human haplotype andis closely related to the protective haplotypes (data not shown).

SNPs IVS2-18insTT and Y402H were also genotyped in 20 unrelated MPGNIIpatients, 52 Rapanui natives and small cohorts of Hispanic Americans,African Americans and European Americans (TABLE 7). The frequency of theat-risk haplotype was estimated in samples from different populationsfrom genotypes of the Y402H variant and/or the IVS10 locus. Theseinclude Rapanui natives over the age of 65 (AMD is extremely rare, andmost likely absent, in this Easter Island population), controls (>65years of age) from Columbia University, Hispanics general population,controls (>65 years of age) from the University of Iowa, AfricanAmericans general population, AMD cases from Columbia University,European Americans general population, AMD cases from the University ofIowa and individuals with MPGNII. N=number of individuals. In the MPGNIIcohort, the frequency of the risk haplotype is approximately 70%. Inaddition, the risk haplotype appears to be lower in frequency inHispanic Americans and African Americans (35-45%), groups with lowerincidences of AMD. However, the number of samples typed in thesepopulations is small. The Rapa Nui population on Easter Island has aremarkably low level of AMD. From the analysis of 52 AMD-free Rapanuinatives over the age of 65, we estimate the frequency of the riskhaplotype to be only 19%.

Discussion Factor H Polymorphisms and AMD

The data presented here links a major proportion of AMD cases in twoindependent cohorts to specific polymorphisms in the complementregulatory gene, Factor H(HF1/CFH) (Zipfel, 2001; Rodriguez de Cordobaet al., 2004). Haplotype analysis shows the most frequent at-riskhaplotype to be present in almost half of individuals with AMD, comparedto approximately 25% of controls. The frequencies and extent of SNPassociations are very similar in the two cohorts and, genotyped SNPsshow highly significant association with AMD in each. The associationsare especially prominent in cases of early AMD or choroidalneovascularization, less so for geographic atrophy. The magnitude of theobserved association of specific HF1 haplotypes with AMD is strikingwhen compared to those genetic abnormalities previously linked to AMD.

Further support for the conclusion that specific HF1 haplotypes conferincreased risk for an AMD disease phenotype was obtained by genotypingof SNPs IVS2-18insTT and Y402H in 20 unrelated patients with MPGNII, arenal disease associated with HF1 mutations in which patients developearly onset macular drusen, and in 52 Rapanui natives, a race with aremarkably low incidence of AMD, if any. Approximately 70% of MPGN IIpatients and 19% of Rapanui were found to harbor the HF1 at-riskhaplotype in this study. Analysis of larger sample sets will be requiredto confirm these results, but the data do suggest that the HF1association with AMD may not be restricted to European-derivedpopulations. Protective haplotypes we also identified, furtherimplicating HF1 function in the pathogenetic mechanisms underlying AMD.

Functional Implications of Factor H Polymorphisms

Factor H deficiencies in humans are associated with MPGN II and atypicalhemolytic uremic syndrome (aHUS) (Zipfel et al., 2001). HF1 deficiencyarises from mutations leading to protein truncations or amino acidsubstitutions that result in protein retention in the endoplasmicreticulum. Reduced levels of plasma HF1 ensue, leading to uncontrolledactivation of the alternative complement pathway with concomitantconsumption of C3 and other complement components. The HF1 mutationsthat lead to aHUS, in contrast, are typically missense mutations thatlimit the complement-inhibitory functions of FH1 at the cell surface.Recent studies have revealed an association between three common SNPsand aHUS in individuals both with and without FH1 mutations (Caprioli etal., 2003). Furthermore, insults such as infection have been documentedto trigger the manifestation of aHUS.

Most of the genotyped SNPs of HF1 are located within importantfunctional domains of the encoded protein (FIG. 3), which consists of 20short consensus repeats (SCR) of 60 amino acids. The SCRs containbinding sites for C3b (SCR1-4, SCR12-14 and SCR19-20), heparin, sialicacid (SCR7, SCR13 and SCR19-20) and C-reactive protein (CRP) (SCR7).Therefore, SNPs located within the functional domains, although common,presumably affect protein function through variability in expressionlevels, binding efficiency, and other molecular properties. For example,the exon 2 I62V variant is located in SCR2, which is included in thefirst C3b binding site, and the exon 9 Y402H variant is within SCR7domain, which binds both heparin and CRP. Intronic SNPs, such as theIVS2-18insTT variant, can affect splicing. For example, the analysis ofthe effect of the TT insertion (on the https://splice.cmh.edu/server)suggested a creation of a new cryptic splice acceptor 6 bp upstream ofthe natural acceptor site (data not shown). It is also possible thatsome of the studied SNPs affect the expression of HF1 isoforms. Forexample, 162V is present in a predicted exon splice enhancer (Wang etal., 2004) (data not shown).

The functional consequences of common SNPs may be modest, since they areinvolved in late-onset phenotypes and not subjected to (rigorous)evolutionary constraint. HF1 variants with more substantial effects,i.e., disease-causing mutations, are implicated in early-onset, severe(recessive) diseases, such as HF1 deficiency and aHUS (Zipfel et al.,2002; Rodriguez de Cordoba et al., 2004; Caprioli at al., 2003; Zipfel,2001). Of interest is the fact that true disease-causing mutations havebeen identified in only about 25% to 35% of HUS patients after completescreening of the HF1 gene (Caprioli et al., 2003). At the same time, adisease-associated haplotype defined by variants −257C>T (promoter),A473A (exon 13) and D936E (exon 18) has been identified in HUS patients,predominantly in those persons with no disease-causing mutations(Caprioli et al., 2003). Moreover, the same study identified severalfamilies where affected probands had inherited a mutated allele from oneparent and a susceptibility allele, from another. By contrast, healthysiblings of affected probands, carriers of the disease-causing mutation,had inherited a protective allele. In affected individuals from thesefamilies a disease-triggering effect, specified as bacterial or viralinfection in >60% of cases, was identified in >80% of all cases andin >90% of cases with no apparent disease-associated mutation (Caprioliet al., 2003).

Together, these data strongly suggest that an at-risk HF1 haplotype incombination with an infectious triggering event is sufficient fordisease manifestation. Interestingly, the at-risk HF1 haplotype in HUS(mainly C-terminal) does not overlap with that in AMD and/or MPGNII(TABLE 2), suggesting different triggers in HUS as opposed to MPGNII andAMD. Observation of early-onset drusen in MPGNII and those of the samecomposition in AMD, but not in HUS, support a different etiology forthese diseases.

Disease-causing mutations in HF1 are rare in MPGN II and have not beenreported in AMD, nor did we find them after extensive screening in thisstudy. However, we observed the same at-risk haplotype at a frequency of70% of patients with MPGN II and approximately 50% in patients with AMD.These data are consistent with an at-risk HF1 haplotype that, iftriggered by an infectious agent, substantially increase one'ssusceptibility to disease. The combined effect of these factorsdetermines the severity the resulting phenotype, ranging from AMD toMPGNII.

Evolutionary analysis of the HF1 haplotype indicates that the riskhaplotype has evolved significantly from the ancestral haplotype foundin the chimpanzee. FIG. 4 depicts a haplotype network diagram of HF1SNPs which shows the relationship between the haplotypes and the size ofthe circles is proportional to the frequency of the haplotype. Thelargefilled-in circle represents the major risk haplotype, thevertically-lined circles are the two significant protective haplotypes,and the large open circle is the haplotype that contains the three SNPsassociated with atypical hemolytic uremic syndrome (HUS), which isneutral for AMD risk. The putative ancestral haplotype is alsoindicated. It is possible that different forms of the HF1 gene emergedin response to pathogens that activate the alternative complementpathway. Weakly acting HF1 haplotypes could provide reduced complementinhibition and stronger protection against bacterial infection. However,such weak alleles could have the consequence of predisposing individualsto the consequences of complement system dysfunction. It is interestingthat the AMD risk haplotype is principally different in the 5′ end ofthe gene that produces both the full length HF1 and the FHL1 protein. Incontract the HUS mutations cluster in the 3′ portion of the gene that isfound only in HF1. Therefore, it will be important to determine the roleof these two forms of the protein in disease.

Biological Model of Factor H Dysfunction in AMD

One of the primary functions of the complement system is to providedefense against such infectious agents. It mediates the opsonization andlysis of microorganisms, removal of foreign particles, recruitment ofinflammatory cells, regulation of antibody production, and eliminationof immune complexes (Morgan et al., 1991; Kinoshita, 1991). Activationof the system triggers a sequential, amplifying, proteolytic cascadethat gives rise to modifications of activating surfaces, to the releaseof soluble anaphylatoxins that stimulate inflammatory cells, andultimately, to formation of the membrane attack complex (MAC), amacromolecular complex that promotes cell lysis through the formation oftransmembrane pores. Uncontrolled activation of complement can lead tobystander damage to host cells and tissues. As a result HF1, as well asother circulating and membrane-associated proteins, have evolved tomodulate the system (Morgan, 1999).

A spectrum of complement components have been identified either withindrusen (and/or the RPE cells that flank or overlie them), along Bruch'smembrane, and/or on the choroidal endothelial cell membrane (Hageman etal., 2001; Mullins et al., 2000; Mullins et al., 2001; Anderson et al.,2002; Johnson, et al., 2000; Johnson et al., 2001; Crabb et al., 2002;Johnson et al., 2002; Mullins et al., 1997). These include terminalpathway complement components, activation-specific fragments of theterminal pathway, as well as various complement modulators. There isevidence that cell-mediated events may also contribute to this process(Penfold et al., 2001; Seddon et al., 2004; Miller et al., 2004).

We now show that HF1 is also a constituent of drusen in human donorswith a prior history of AMD. Secondly, we show that HF1 co-localizeswith its ligand C3b in amyloid-containing substructural elements withindrusen, further implicating these structures as candidate complementactivators (Anderson et al., 2004; Johnson et al., 2002). We alsodemonstrate that HF1 and the MAC, as shown by C5b-9 immunoreactivity,co-distribute at the RPE-choroid interface and that these deposits aremore robust in eyes from donors with prior histories of AMD. Finally,HF1 and C5b-9 immunoreactivities are more intense in the macula comparedto more peripheral locations from the same eye. All of these findingsare consistent with the fact that AMD pathology is manifested primarilyin the macula and with the conclusion that complement activation at thelevel of Bruch's membrane is a key element in the process of drusenformation as well as a contributing factor in the pathogenesis of AMDHageman et al., 2001; Anderson et al., 2002).

The distribution of HF1 at the RPE-choroid interface is strikinglysimilar to that of C5b-9, implying that significant amounts of the MACare generated and deposited at the RPE-choroid interface. This suggeststhat the protein associated with the at-risk HF1 haplotype(s) mayundergo a reduction in its normal ability to attenuate complementactivation. Thus, the HF1 variants associated with AMD may put RPE andchoroidal cells at sustained risk for alternative pathway-mediatedcomplement attack, drusen formation and the disruption of Bruch'smembrane integrity that is associated with late-stage neovascular AMD.Since Bruch's membrane is significantly thinner in the macula thanelsewhere (Chong et al., 2005), it may be more susceptible to subsequentneovascular invasion. Because Bruch's membrane is significantly thinnerin the macula than in the periphery, it may be more likely to becomedegraded to an extent that it is susceptible to neovascular invasion.

In summary, the results of this investigation provide strong evidencethat common haplotypes in HF1 predispose individuals to AMD. We proposethat alterations in genes that regulate the alternative pathway of thecomplement cascade, in combination with events that activate the system,underlie a major proportion of AMD in the human population.

Example 2 Variations in the Complement Regulatory Genes Factor H(CFH)and Factor H Related 5 (CFHR5) are Associated with MembranoproliferativeGlomerulonephritis Type II (Dense Deposit Disease) Introduction

The membranoproliferative glomerulonephritides are diseases of diverseand often obscure etiology that account for 4% and 7% of primary renalcauses of nephrotic syndrome in children and adults, respectively (Orthet al., 1998). Based on renal immunopathology and ultrastructuralstudies, three subtypes are recognized. Membranoproliferativeglomerulonephritis (MPGN) types I and III are variants of immunecomplex-mediated disease; MPGNII, in contrast, has no known associationwith immune complexes (Appel et al., 2005).

MPGNII accounts for less than 20% of cases of MPGN in children and onlya fractional percentage of cases in adults (Orth et al., 1998; Habib etal., 1975; Habib et al., 1987). Both sexes are affected equally, withthe diagnosis usually made in children between the ages of 5-15 yearswho present with non-specific findings like hematuria, proteinuria,acute nephritic syndrome or nephrotic syndrome (Appel et al., 2005).More than 80% of patients with MPGNII are also positive for serum C3nephritic factor (C3NeF), an autoantibody directed against C3bBb, theconvertase of the alternative pathway of the complement cascade(Schwertz et al., 2001). C3NeF is found in up to one-half of personswith MPGN types I and III and also in healthy individuals, making theelectron microscopic demonstration of dense deposits in the glomerularbasement membrane (GBM) necessary for a definitive diagnosis of MPGN II(Appel et al., 2005). This morphological hallmark is so characteristicof MPGN II that the disease is more accurately referred to as DenseDeposit Disease (MPGNII/DDD) (FIG. 12).

Spontaneous remissions of MPGNII/DDD are uncommon (Habib et al., 1975;Habib et al., 1987; Cameron et al., 1983; Barbiano di Belgiojoso et al.,1977). The more common outcome is chronic deterioration of renalfunction leading to end-stage renal disease (ESRD) in about half ofpatients within 10 years of diagnosis (Barbiano di Belgiojoso et al.,1977; Swainson et al., 1983). In some patients, rapid fluctuations inproteinuria occur with episodes of acute renal deterioration in theabsence of obvious triggering events; in other patients, the diseaseremains stable for years despite persistent proteinuria.

C3NeF persists throughout the disease course in more than 50% ofpatients with MPGNII/DDD (Schwertz et al., 2001). Its presence istypically associated with evidence of complement activation, such as areduction in CH50, decrease in C3, increase in C3dg/C3d and persistentlyhigh levels of activation of the alternative pathway of the complementcascade. C3, the most abundant complement protein in serum (˜1.2 mg/ml),normally undergoes low levels of continuous autoactivation by hydrolysisof its thioester in a process known as tick-over. C3 hydrolysis inducesa large conformational protein change, making C3(H₂O) similar to C3b, acleavage product of C3. C3(H₂O) associates with factor B to formC3(H₂O)Bb, which cleaves C3 to C3b in an amplification loop thatconsumes C3 and produces C3bBb² (FIG. 13).

In MPGNII/DDD, C3NeF binds to C3bBb (or to the assembled convertase) toprolong the half-life of this enzyme, resulting in persistent C3consumption that overwhelms the normal regulatory mechanisms to controllevels of C3bBb and complement activation (Appel et al., 2005). Normalcontrol involves at least seven proteins, four of which are present inserum (complement Factor H(CFH), complement factor H-like protein 1(CFHL1), complement factor I (CFI) and C4 binding protein (C4BP)) andthree of which are cell membrane-associated (membrane co-factor protein(MCP, CD46), decay accelerating factor (DAF, CD55) and complementreceptor 1 (CR1, CD35)) (Appel et al., 2005; Meri et al., 1994; Pascualet al., 1994).

Of particular relevance to MPGNII/DDD is Factor H, one of 7 proteins inthe Factor H family. In pigs and mice, its deficiency is associated withthe development of renal disease that is similar at the light andelectron microscopic level to MPGNII/DDD, and in humans its deficiencyas well as mutations in the Factor H gene have been reported in patientswith MPGNII/DDD (Meri et al., 1994; Dragen-Durey et al., 2004; Zipfel etal., 2005) (FIG. 14).

The other 6 members of the Factor H family include FHL1, which is asplice isoform of Factor H, and five CFH-related proteins encoded bydistinct genes (CFHR1-5). There is little known about the latter fiveproteins, although they do show varying degrees of structural similarityto Factor H (Appel et al., 2005). Most interesting in this group withrespect to MPGNII/DDD is CFHR5, because it shows the highest similarityto Factor H and has been demonstrated in renal biopsies of patients withother types of glomerulonephritis (Appel et al., 2005; Murphy et al.,2002). In vitro studies have also shown that V is present on surfacesexposed to complement attack, suggesting a possible role in thecomplement cascade (Murphy et al., 2002).

A possible relationship between Factor H/CFHR5 and MPGNII/DDD is furtherstrengthened by the observation that patients with MPGNII/DDD develop anocular phenotype called drusen. Drusen result from the deposition ofabnormal extracellular deposits in the retina within the ocular Bruch'smembrane beneath the retinal pigment epithelium. The drusen ofMPGNII/DDD are clinically and compositionally indistinguishable fromdrusen that form in age-related macular degeneration (AMD) (Mullins etal., 2001; Anderson et al., 2002), which is the most common form ofvisual impairment in the elderly (Klein et al., 2004; van Leeuwen etal., 2003). The single feature that distinguishes these two types ofdrusen is age of onset-drusen in MPGNII/DDD develop early, often in thesecond decade of life, while drusen in AMD are found in the elderly.

Four recent studies have implicated specific allele variants of Factorwith AMD, suggesting that subtle differences in Factor H-mediatedregulation of the alternative pathway of complement may play a role in asubstantial proportion of AMD cases (Hageman et al., 2005; Edwards etal., 2005; Haines et al., 2005; Klein et al., 2005). One of thesestudies also showed that MPGNII/DDD and AMD patients segregate severalof the same Factor H risk alleles (Hageman et al., 2005). In this study,we sought to refine the association of allele variations of Factor H andCFHR5 with MPGNII/DDD.

Materials and Methods

Patients and Controls. Patients with biopsy-proven MPGNII/DDD wereascertained in nephrology divisions and enrolled in this study underIRB-approved guidelines. The control group was ascertained fromethnically-matched but not age-matched unrelated persons in whom AMD hadbeen excluded by opthalmologic examination.

Mutation Screening and Analysis. Coding and adjacent intronic regions ofFactor H and CFHR5 were PCR amplified for 35 cycles of 30 seconds eachat 94° C. denaturing, 61° C. annealing and 70° C. extension. Thesequences of primers used to amplify the Factor H and CFHR5 codingsequences are shown in TABLES 10 and 11, respectively. Productgeneration was verified by agarose gel electrophoresis and ampliconswere then bi-directionally sequenced in patients with MPGNII/DDD. Allnovel and reported SNPs identified through data mining (Ensembledatabase, dbSNP, Applied Biosystems) were typed in the controlpopulation by denaturing high performance liquid chromatography (DHPLC)(Tables 9 and 10). In brief, DHPLC analysis of each amplicon wasperformed at three different temperatures. Amplicons were analyzed usingWavemaker software to estimate optimal temperature, run time andacetonitrile gradient. Predicted temperatures were bracketed by +/−2° C.to optimize sensitivity and maximize the likelihood that novel mutationswould be detected (Prasad et al., 2004).

Haplotype Analysis. Construction of block structures with distributionof haplotypes was completed using Haploview, a publicly availablesoftware program developed at the Whitehead Institute(http://www.broad.mit.edu/mpg/haploview/) (see Barrett et al.). Twodatasets, one consisting of each control's sex and genotype, and theother describing marker information including SNP identification andchromosomal location, were assimilated in Excel files, which wereup-loaded into the Haploview program. The output consisted of linkagedisequilibrium (LD) plots and the corresponding population frequencieswith crossover percentages.

Statistical Analysis. The chi-square test of independence was used todetect differences in SNP frequencies between patients with MPGNII/DDDand controls. P-values≦0.05 were considered significant. The LD plotsfor Factor H and CFHR5 were created using the control population.

Results

Patients and Controls. Twenty-two patients with biopsy-proven MPGNII/DDDand 131 persons without AMD participated in this study. Mean age of thecontrol group was 78.4 years, reflecting our ascertainment criterion toexclude AMD.

Factor H, CFHR5 and MPGNII/DDD. Allele frequencies of four of sevenFactor H SNPs genotyped in the MPGNII/DDD patient group and the controlpopulation showed a significant association with the MPGNII/DDD diseasephenotype at p<0.05. These SNPs included exon 2 I62V, IVS 2-18insTT,exon 9 Y402H and exon 10 A473A. Allele frequencies for exon 7 A307A,exon 13 Q672Q and exon 18 D936E were not significantly different betweengroups (TABLES 11 to 13).

Five CFHR5 SNPs were genotyped in the MPGNII/DDD patient group andcontrol population, including one non-synonymous SNP (exon 2 P46S), twopromoter SNPs (−249T>C, −20 T>C) and two intronic SNPs (IVS1+75T>A,IVS2+58C>T). Allele frequencies of three SNPs—exon 2 P46S, −249T>C and−20 T>C—were significantly different between groups at p<0.05 (TABLES 14and 15).

Haploblocks. Haplotype blocks showed that A307A and Y402H are in linkagedisequilibrium in Factor H while −249T>C and −20T>C are in linkagedisequilibrium in CFHR5 (FIG. 15).

Discussion

The alternative pathway of complement represents an elegant system toprotect humans from pathogens. Its central component, C3, circulates ata high concentration in plasma and is distributed throughout body fluids(Walport, 2001). Its activation creates a toxic local environment thatdamages foreign surfaces and results in the elimination of microbes. Toprevent unrestricted complement activation, host cells and tissuesurfaces down-regulate the amplification loop using a combination ofsurface-attached and membrane-bound regulators of complement. Some hostcells express a single membrane-bound regulator of complement in highcopy number, while other cells express several membrane-bound regulatorsand also attach soluble fluid-phase regulators. A few tissues lackmembrane-bound regulators and depend exclusively on the attachment ofsoluble regulators (Appel et al., 2005).

In the kidney, endothelial and mesangial cells express twomembrane-bound regulators of complement, MCP and DAF (van denDobbelsteen et al., 1994; Timmerman et al., 1996). Podocytes expressfour: MCP, DAF, CR1 and CD59. Both mesangial cells and podocytes alsosecrete the soluble regulator, Factor H, which is up-regulated inmembranous nephropathy in response to complement activation andinflammation (Angaku et al., 1998; Bao et al., 2002). Factor H acts inan autocrine fashion by binding directly to the secreting mesangialcells and podocytes.

The GBM, in contrast, is unique. It lacks endogenous membrane-boundregulators to protect it from complement-mediated injury, however itshighly negatively charged surface binds and absorbs Factor H (Zipfel etal., 2005). The dependency of the GBM on Factor H for local complementcontrol is consistent with the finding of pathologic mutations in FactorH in a few persons with MPGNII/DDD (Ault et al., 1997; Dragen-Durey etal., 2004).

Our data identifying several allele variants of Factor H and CFHR5associated with MPGNII/DDD is consistent with the hypothesis thatcomplement control plays a role in the pathogenesis of this disease. Acomparison of our data with reported AMD data adds additional support,as the allele frequency for each of the identified at-risk SNP variantswe observed in Factor H is higher in the MPGNII/DDD patient cohort thanin the AMD patient cohort, and strong evidence implicates Factor H inAMD (Hageman et al., 2005; Edwards et al., 2005; Haines et al., 2005;Klein et al., 2005). Although it is not known whether the amino acidchanges in exons 2 and 9 of Factor H impact function, these changes arefound in domains that interact with C3b and heparin, and differences inC3b/C3d and heparin binding have been demonstrated with several aminoacid changes in Factor H that are associated with another renal disease,atypical hemolytic uremic syndrome (Manuelian et al., 2003) (TABLES 12and 13).

With the exception of Factor H, the function of other members of theFactor H-related family is largely unknown and their expression patternshave not been explored, however studies of CFHR5 have shown that it hasproperties similar to Factor H, including heparin, CRP and C3b binding(Murphy et al., 2002) (FIG. 14). This similarity suggests that likeFactor H, CFHR5 plays a role in MPGNII/DDD. Consistent with this is ourfinding of CFHR5 expression in renal biopsies from two patients withMPGNII/DDD (data not shown).

Our genotyping data show that some allele variants of CFHR5preferentially associate with the MPGNII/DDD disease phenotype. Includedare two SNPs in the promoter region of CFHR5 which could affecttranscription, one by removing a binding site for C/EBPbeta and theother by adding a GATA-1 binding site. The other significant associationchanges a proline to serine in exon 2. Since exons 1 and 2 of CFHR5encode a domain homologous to short consensus repeat 6 (SCR6) of FactorH, which is integral to heparin and CRP binding, this change couldaffect complement activation and control.

Example 3 Production of Protective Form of Factor H Protein

An exemplary protective form of human complement factor H(CFH) wasprepared based on haplotype H2 (FIG. 5). Briefly, RNA was isolated fromocular tissues (RPE/choroid complexes) of four donors. The RNA wasamplified by reverse transcription-polymerase chain reaction using thefollowing primers:

5′-GAAGATTGCAATGAACTTCCTCCAAG-3′ [SEQ ID NO:331]5′-AAGTTCTGAATAAAGGTGTGC-3′. [SEQ ID NO:332]RT-PCR reactions were performed using Superscript III One-Step HighFidelity with Platinum Taq, as described by the manufacturer(Invitrogen, Carlsbad, Calif.). The appropriate sized products (3,769bp) were excised from agarose gels and isolated using spin columns.

The PCR products were cloned using the TOPO-TA cloning system, asrecommended by the manufacturer (Invitrogen) in the vector pCR2.1-TOPO.The complete genetic sequences of the clones derived from each of thefour patients were determined by direct sequencing. The DNA derived fromone patient (patient #498-01) had the fewest number of nucleotidepolymorphisms relative to that of an exemplary protective referencesequence (H2), although this DNA encoded the risk sequence at amino acidposition 402 (histidine) and encoded valine at amino acid position 62.To prepare a gene encoding a protective form of CFH we changed the basesencoding amino acid 62 such that it coded for isoleucine and those atposition 402 such that they encoded a tyrosine, using the QuikChangeMutagenesis system (Stratagene, La Jolla, Calif.), resulting in SEQ IDNO:335. The amino acid at position 1210 of this protein is arginine. Theoligonucleotides employed were as follows (plus the appropriateantisense version):

62: [SEQ ID NO:333] 5′-TATAGATCTCTTGGAAATATAATAATGGTATGCAGG-3′ 402: [SEQID NO:334] 5′-ATGGATATAATCAAAATTATGGAAGAAAGTTTGTAC-3′The fidelity of the introduced mutations were confirmed by directsequencing of the entire gene. The resulting protective gene was clonedinto the eukaryotic expression vector pcDNA3.1 (Invitrogen) undercontrol of the cytomegalovirus promoter. This expression vector wastransfected into the human lung carcinoma cell line A549 (ATCC,Manassas, Va.) using the Exgen 500 transfection reagent (Fermentas,Hanover, Md.). Subsequent to transfection, cells were grown inserum-free media (Hybridoma-SFM, Invitrogen).

Supernatants were collected 48 hours after transfection and subjected toWestern blot analyses. The presence of the appropriate-sized product(approximately 150 kDa) was confirmed using monoclonal and polyclonalantibodies directed against human CFH (Quidel, San Diego, Calif.).

Patient #498-01 (62I, 402Y) CFH Gene [SEQ ID NO:335]AGTTAGCTGGTAAATGTCCTCTTAAAAGATCCAAAAAATGAGACTTCTAGCAAAGATTATTTGCCTTATGTTATGGGCTATTTGTGTAGCAGAAGATTGCAATGAACTTCCTCCAAGAAGAAATACAGAAATTCTCACAGGTTCCTGGTCTGACCAAACATATCCAGAAGGCACCCAGGCTATCTATAAATGCCGCCCTGGATATAGATCTCTTGGAAATATAATAATGGTATGCAGGAAGGGAGAATGGGTTGCTCTTAATCCATTAAGGAAATGTCAGAAAAGGCCCTGTGGACATCCTGGACATACTCCTTTTGGTACTTTTACCCTTACAGGAGGAAATGTGTTTGAATATGGTCTAAAAGCTGTGTATACATGTAATGAGGGGTATCAATTGCTAGGTGAGATTAATTACCGTGAATGTGACACAGATGGATGGACCAATGATATTCCTATATGTGAAGTTCTGAAGTGTTTACCAGTGACAGCACCACAGAATGGAAAAATTGTCAGTAGTGCAATGGAACCAGATCGGGAATACCATTTTGGACAAGCAGTACGGTTTGTATGTAACTCAGGCTACAAGATTGAAGGAGATGAAGAAATGCATTGTTCAGACGATGGTTTTTGGAGTAAAGAGAAACCAAAGTGTGTGGAAATTTCATGCAAATCCCCAGATGTTATAAATGGATCTCCTATATCTCAGAAGATTATTTATAAGGAGAATGAACGATTTCAATATAAATGTAACATGGGTTATGAATACAGTGAAAGAGGAGATGCTGTATGCACTGAATCTGGATGGCGTCCGTTCCCTTCATGTGAAGAAAAATCATGTGATAATCCTTATATTCCAAATGGTGACTACTCACCTTTAAGGATTAAACACAGAACTGGAGATGAAATCACGTACCAGTGTAGAAATGGTTTTTATCCTGCAACCCGGGGAAATACAGCAAAATGCACAAGTACTGGCTGGATACCTGCTCCGAGATGTACCTTGAAACCTTGTGATTATCCAGACATTAAACATGGAGGTCTATATCATGAGAATATGCGTAGACCATACTTTCCAGTAGCTGTAGGAAAATATTACTCCTATTACTGTGATGAACATTTTGAGACTCCGTCAGGAAGTTACTGGGATCACATTCATTGCACACAAGATGGATGGTCGCCAGCAGTACCATGCCTCAGAAAATGTTATTTTCCTTATTTGGAAAATGGATATAATCAAAATTATGGAAGAAAGTTTCTACAGGGTAAATCTATAGACGTTGCCTGCCATCCTGGCTACGCTCTTCCAAAAGCGCAGACCACAGTTACATGTATGGAGAATGGCTGGTCTCCTACTCCCAGATGCATCCGTGTCAAAACATGTTCCAAATCAAGTATAGATATTGAGAATGGGTTTATTTCTGAATCTCAGTATACATATGCCTTAAAAGAAAAAGCGAAATATCAATGCAAACTAGGATATGTAACAGCAGATGGTGAAACATCAGGATCAATTACATGTGGGAAAGATGGATGGTCAGCTCAACCCACGTGCATTAAATCTTGTGATATCCCAGTATTTATGAATGCCAGAACTAAAAATGACTTCACATGGTTTAAGCTGAATGACACATTGGACTATGAATGCCATGATGGTTATGAAAGCAATACTGGAAGCACCACTGGTTCCATAGTGTGTGGTTACAATGGTTGGTCTGATTTACCCATATGTTATGAAAGAGAATGCGAACTTCCTAAAATAGATGTACACTTAGTTCCTGATCGCAAGAAAGACCAGTATAAAGTTGGAGAGGTGTTGAAATTCTCCTGCAAACCAGGATTTACAATAGTTGGACCTAATTCCGTTCAGTGCTACCACTTTGGATTGTCTCCTGACCTCCCAATATGTAAAGAGCAAGTACAATCATGTGGTCCACCTCCTGAACTCCTCAATGGGAATGTTAAGGAAAAAACGAAAGAAGAATATGGACACAGTGAAGTGGTGGAATATTATTGCAATCCTAGATTTCTAATGAAGGGACCTAATAAAATTCAATGTGTTGATGGAGAGTGGACAACTTTACCAGTGTGTATTGTGGAGGAGAGTACCTGTGGAGATATACCTGAACTTGAACATGGCTGGGCCCAGCTTTCTTCCCCTCCTTATTACTATGGAGATTCAGTGGAATTCAATTGCTCAGAATCATTTACAATGATTGGACACAGATCAATTACGTGTATTCATGGAGTATGGACCCAACTTCCCCAGTGTGTGGCAATAGATAAACTTAAGAAGTGCAAATCATCAAATTTAATTATACTTGAGGAACATTTAAAAAACAAGAAGGAATTCGATCATAATTCTAACATAAGGTACAGATGTAGAGGAAAAGAAGGATGGATACACACAGTCTGCATAAATGGAAGATGGGATCCAGAAGTGAACTGCTCAATGGCACAAATACAATTATGCCCACCTCCACCTCAGATTCCCAATTCTCACAATATGACAACCACACTGAATTATCGGCATGGAGAAAAAGTATCTGTTCTTTGCCAAGAAAATTATCTAATTCAGGAAGGAGAAGAAATTACATGCAAAGATGGAAGATGGCAGTCAATACCACTCTGTGTTGAAAAAATTCCATGTTCACAACCACCTCAGATAGAACACGGAACCATTAATTCATCCAGGTCTTCACAAGAAAGTTATGCACATGGGACTAAATTGAGTTATACTTGTGAGGGTGGTTTCAGGATATCTGAAGAAAATGAAACAACATGCTACATGGGAAAATGGAGTTCTCCACCTCAGTGTGAAGGCCTTCCTTGTAAATCTCCACCTGAGATTTCTCATGGTGTTGTAGCTCACATGTCAGACAGTTATCAGTATGGAGAAGAAGTTACGTACAAATGTTTTGAAGGTTTTGGAATTGATGGGCCTGCAATTGCAAAATGCTTAGGAGAAAAATGGTCTCACCCTCCATCATGCATAAAAACAGATTGTCTCAGTTTACCTAGCTTTGAAAATGCCATACCCATGGGAGAGAAGAAGGATGTGTATAAGGCGGGTGAGCAAGTGACTTACACTTGTGCAACATATTACAAAATGGATGGAGCCAGTAATGTAACATGCATTAATAGCAGATGGACAGGAAGGCCAACATGCAGAGACACCTCCTGTGTGAATCCGCCCACAGTACAAAATGCTTATATAGTGTCGAGACAGATGAGTAAATATCCATCTGGTGAGAGAGTACGTTATCAATGTAGGAGCCCTTATGAAATGTTTGGGGATGAAGAAGTGATGTGTTTAAATGGAAACTGGACGGAACCACCTCAATGCAAAGATTCTACAGGAAAATGTGGGCCCCCTCCACCTATTGACAATGGGGACATTACTTCATTCCCGTTGTCAGTATATGCTCCAGCTTCATCAGTTGAGTATCAATGCCAGAACTTGTATCAACTTGAGGGTAACAAGCGAATAACATGTAGAAATGGACAATGGTCAGAACCACCAAAATGCTTACATCCGTGTGTAATATCCCGAGAAATTATGGAAAATTATAACATAGCATTAAGGTGGACAGCCAAACAGAAGCTTTATTCGAGAACAGGTGAATCAGTTGAATTTGTGTGTAAACGGGGATATCGTCTTTCATCACGTTCTCACACATTGCGAACAACATGTTGGGATGGGAAACTGGAGTATCCAACTTGTGCAAAAAGATAGAATCAATCATAAAGTGC ACACCTTTATTCAGAACTT

TABLE 1A dbSNP SEQ ID AA Allele Freq. Allele Freq. Chi2 & No. LocationSequence Spanning Polymorphism No: Change CTL AMD P Value Promoter 1GGGGTTTTCTGGGATGTAAT  9 1-0.944:2-0.056 1-0.96:2-0.04   [A/G]ATGTTCAGTGTTTTG ACCTT CCCCAAAAGACCCTACATTA [T/C]TACAAGTCACAAAACTGGAA rs3753394 Promoter 4 TTATGAAATCCAGAGGATAT 10 1-0.31:2-0.691-0.25:2-0.75 6.485:0.039 [C/T]ACCAGCTGCTGATTT GCACAAATACTTTAGGTCTCCTATA [G/A]TGGTCGACGACTAAA CGTGT rs529825 Intron 1AGTCCAAGTTTACACAGTAC 11 1-0.74:2-0.26 1-0.84:2-0.16 26.07:2.18E−06[G/A]ATAGACTTACCCATT GCCAA TCAGGTTCAAATGTGTCATG [C/T]TATCTGAATGGGTAACGGTT rs800292 Exon 2 GATATAGATCTCTTGGAAAT 12 I62V 1-0.76:2-0.221-0.91:2-0.09 16.19:5.74E−05 [G/A]TAATAATGGTATGCA GGAAGCTATATCTAGAGAACCTTTA [C/T]ATTATTACCATACGT CCTTC Intron 2TAATTCATAACTTTTTTTTT 13 1-0.77:2-0.23 1-0.89:2-0.11 22.19:2.47E−06[-/TT]CGTTTTAGAAAGGC CCTGTG ATTAAGTATTGAAAAAAAAA [-/AA]GCAAAATCTTTCCGGGACAC rs3766404 Intron 6 AAAGGAATACATTTAGGACT 14 1-0.83:2-0.171-0.91:2-0.09 23.82:671E−06 [C/T]ATTTGAAGTTAGTGT CAACATTTCCTTATGTAAATCCTGA [G/A]TAAACTTCAATCACA GTTGT rs1061147 Exon 7CAACCCGGGGAAATACAGC 15 A307A 1-0.34:2-0.66 1-0.59:2-0.41 50.39:1.26E−12[A/C]AAATGCACAAGTACT GGCTG GTTGGGCCCCTTTATGTCG [T/G]TTTACGTGTTCATGACCGAC rs1061170 Exon 9 AAAATGGATATAATCAAAAT 16 Y402H 1-0.66:2-0.341-0.46:2-0.54 55.20:1.03E−12 [T/C]ATGGAAGAAAGTTTG TACAGTTTTACCTATATTAGTTTTA [A/G]TACCTTCTTTCAAAC ATGTC rs2274700 Exon 10TATGCCTTAAAAGAAAAAGC 17 A473A 1-0.54:2-0.46 1-0.80:2-0.20 36.48:1.55E−09[G/A]AAATATCAATGCAAA CTAGG ATACGGAATTTTCTTTTTCG [C/T]TTTATAGTTACGTTTGATCC Exon 10A CAGCTTGAGTGGATCAAAGA 18 1-1.00:2-0.00 1-0.933:2-0.067[-/N]TGACAAGGGCCAATG GAACC GTCGAACTCACCTAGTTTCT [-/N]ACTGTTCCCGGTTACCTTGG rs203674 Intron 10 ACGGTACCTATTTATTAGTA 19 1-0.66:2:0.341-0.44:2-0.56 66.97:286E−15 [G/T]ATCTAATCAATAAAG CTTTTTGCCATGGATAAATAATCAT [C/A]TAGATTAGTTATTTC GAAAA rs203674 Intron 10*AAAAGCTTTATTGATTAGAT 63 1-0.66:2:0.34 1-0.44:2-0.56 66.97:2.86E-15[A/C]TACTAATAAATAGGT ACCGT rs3753396 Exon 13 AAGGGACCTAATAAAATTCA 20Q672Q 1-0.84:2-0.16 1-0.86:2-0.14 0.308:0.579    [A/G]TGTGTTGATGGAGAGTGGAC TTCCCTGGATTATTTTAAGT [T/C]ACACAACTACCTCTC ACCTG rs375046 Intron 15TTTTTTATTTTTTATTATAA 21 1-0.67:2-0.31 1-0.85:2-0.14 [C/A]ATTAATTATATTTTTAATAT AAAAAATAAAAAATAATATT [G/T]TAATTAATATAAAAA TTATA rs1065489 Exon 18CCTTGTAAATCTCCACCTGA 22 D936E 1-0.87:2-0.13 1-0.85:2-0.15 0.155:0.694   [G/T]ATTTCTCATGGTGTT GTAGC GGAACATTTAGAGGTGGACT [C/A]TAAAGAGTACCACAACATCG Exon 22 GGGGATATCGTCTTTCATCA 23 R1210C 1-1.00:2-0.00 1-0095:2-0.05[C/T]GTTCTCACACATTGC GAACA CCCCTATAGCAGAAAGTAGT [G/A]CAAGAGTGTGTAACGCTTGT *Shows the non-coding strand of the genomic sequence.

TABLE 1B (1) SNP name Interrogated Sequence SEQ ID NO: Chimp Location AArs3753394 AAATCCAGAGGATAT[C/T]ACCAGCTGCTGATTT 24 C Promoter rs529825AATGGGTAAGTCTAT[C/T]GTACTGTGTAAACTT 25 T Intron 1 rs800292TGCATACCATTATTA[C/T]ATTTCCAAGAGATCT 26 T Exon 2 I62V Intron 2ACATACTAATTCATAAC[-/TT]TTTTTTTTTCGTTTTAG 27 Intron 2 insTT rs3766404AATACATTTAGGACT[T/C]ATTTGAAGTTAGTGT 28 C Intron 6 rs1061147CCGGGGAAATACAGC[C/A]AAATGCACAAGTACT 29 A Exon 7 A307A rs1061170GGATATAATCAAAAT[T/C]ATGGAAGAAAGTTTG 30 T Exon 9 Y402H rs2274700CTTAAAAGAAAAAGC[G/A]AAATATCAATGCAAA 31 G Exon 10 A473A rs203674CTTTATTGATTAGAT[A/C]TACTAATAAATAGGT 32 A Intron 10 rs3753396ACCTAATAAAATTCA[A/G]TGTGTTGATGGAGAG 33 A Exon 13 Q672Q rS1065489TAAATCTCCACCTGA[G/T]ATTTCTCATGGTGTT 34 G Exon 18 D936E (2) SEQ ID SEQ IDSNP name Forward Primer or AOD number NO: Reverse Primer NO: rs3753394C_2530387_10 rs529825 C_2250476_10 rs800292 C_2530382_10 Intron 2ACTTGTTCCCCCACTCCTAC 35 CCTCTTTTCGTATGGACTAC 36 InsTT rs3766404C_11890065_10 rs1061147 TGAAATCACGTACCAGTGTAGAAATGG 37CAGGTATCCAGCCAGTACTTGT 38 rs1061170 CTTTATTTATTTATCATTGTTATGGTC 39GGCAGGCGACGTCTATAGATTTACC 40 CTTAGGAAAATGTTATTT rs2274700TCACCATCTGCTGTTACATATCCTAGT 41 TGGGTTTATTTCTGAATCTCAGTAT 42 ACATATGCrs203674 C_2530311_10 rs3753396 C_2530296_10 rs1065489 C_2530274_10 (3)SNP name VIC Probe SEQ ID NO: FAM Probe SEQ ID NO: rs1061147AATACAGCAAAATGC 43 ATACAGCCAAATGC 46 rs1001170 TTTCTTCCATGATTTTG 44TTCTTCCATAATTTTG 47 rs2274700 AAGAAAAAGCGAAATAT 45 AAGAAAAAGCAAAATAT 48

TABLE 1C Location Sequence Spanning Polymorphism AA Position SEQ ID NO:Exon 2 CCAGGCTATCTATAAATGCC[G/A]CCCTGGATATAGATCTCTTG R53H 49 Exon 3TTGGTACTTTTACCCTTACA[G/T]GAGGAAATGTGTTTGAATAT G100R 50 Exon 5ACGATGGTTTTTGGAGTAAA[G/notG]AGAAACCAAAGTGTGTGGGT  201 51 Exon 6TTATTATTAAGGAGAATGAA[C/notC]GATTTCAATATAAATGTAAC R232X 52 Exon 6CACTGAATCTGGATGGCGTC[C/notC]GTTGCCTTCATGTGAAG(end Exon 6)  258 53 Exon 8AAGATGGATGGTCGCCAGCA[G/C][-/C]TACCATGCCTCA(end Exon 8) V383L 54 Exon 16ACAATTATGCCCACCTCCAC[C/G]TCAGATTCCCAATTCTCACA  815 55 Exon 17CAACCACCTCAGATAGAACA[C/T]GGAACCATTAATTCATCCAG H878H 56 Exon 17GTCTTCACAAGAAAGTTATG[C/T]ACATGGGACTAAATTGAGTT A892V 57 Exon 18CACATGTCAGACAGTTATCA[G/T]TATGGAGAAGAAGTTACGTA Q950H 58 Exon 18TCAGTATGGAGAAGAAGTTA[C/T]GTACAAATGTTTTGAAGGTT S956L 59 Intron 18 (beginIVS18)GTATGG[G/T]GCATTGAATTTTATTATATG 60 Exon 20 (begin Exon20)ACACCTCCTGTGTG[A/T]ATCCGCCCACAGTACAAAAT N1050Y 61 Exon 21CTTGTATCAACTTGAGGGTA[-/N]A[-/N]CAAGCGAATAACATGTAGAAA 1147 62

TABLE 2 Haplotype Analysis of HF1 SNPs in AMD Cases and Controls Prom  IVS1  I62V     IVS6 Y402H   IVS10 Q672Q D936E OR est P Cases Con

2.67 0.48 0.63 <0.000001 <0.000011  0.00035 0.49 0.06 0.12 0.28 0.120.18

2.68 0.50 0.62 0.48 0.06 0.12 0.26 0.12 0.18

2.65 0.50 0.6 0.49 0.06 0.12 0.26 0.12 0.19

2.56 0.49 0.6 0.49 0.06 0.12 0.27 0.12 0.19

2.33 0.51 0.56 0.61 0.06 0.18 0.31 0.12 0.21

2.54 0.48 0.58 0.51 0.07 0.12 0.29 0.14 0.19

2.55 0.46 0.58 0.51 0.07 0.12 0.29 0.14 0.19

2.46 0.47 0.56 0.51 0.07 0.12 0.30 0.14 0.20

2.47 0.48 0.55 0.51 0.07 0.12 0.30 0.14 0.20

2.12 0.61 0.53 0.76 0.08 0.13 0.60 0.14 0.21

2.49 0.45 0.58 0.52 0.29 0.12 0.30 0.47 0.19

TABLE 3 rs3753394 rs529825 rs3766404 rs203674 rs3753396 rs1065489Promoter Intron 1 Intron 6 Intron 10 Exon 13 Exon 18 Haplotype Freq. CTLFreq. AMD 1 1 1 2 1 1 111211 0.28436 0.478059 1 2 1 1 1 1 1211110.210856 0.131313 2 1 1 1 2 2 211122 0.149247 0.126697 1 1 2 1 1 1112111 0.129893 0.061917 2 1 1 1 1 1 211111 0.094861 0.07125 2 1 1 2 1 1211211 0.046438 0.06834 1 2 2 1 1 1 122111 0.026677 0.014859 2 1 2 1 1 1212111 0.012731 0.01473 1 1 1 1 1 1 111111 0.012686 0.007305 1 2 1 2 1 1121211 0.012684 0.004723 1 2 1 1 2 2 121122 0.009249 0.000945 1 1 1 1 22 111122 0.007487 0.008705 2 1 2 1 2 1 212121 0.002049 1 2 2 2 1 1122211 0.00078 0.000488 2 1 2 1 2 2 212122 0.000001 2 1 1 2 1 2 2112120.002175 2 1 1 1 1 2 211112 0.001869 2 2 1 1 1 1 221111 0.00169 2 1 2 21 1 212211 0.001061 1 2 1 2 2 2 121222 0.00096 1 1 2 1 1 2 1121120.00095 2 1 1 1 2 1 211121 0.00095 2 1 2 2 1 2 212212 0.00069 2 2 1 2 11 221211 0.000322 1 2 2 1 1 2 122112 0.000001

TABLE 4 HF1 SNP Association with Age-related Macular DegenerationPromoter IVS1 Exon 2 IVS2 IVS6 rs3753394 rs529825 I62V insTT rs3766404Iowa # Controls 68 126 # Cases 228 390 X2 15 22.21 P 0.000108 2.44 ×10⁻⁰⁶ OR 2.79 2.38 95% CI 1.67-4.65 1.65-3.44 Columbia # Controls 126266 261 273 271 # Cases 329 547 546 549 546 X2 8.61 25.4 36.12 28.423.04 P 0.00334 4.66 × 10⁻⁰⁷ 3.21 × 10⁻⁰⁷ 9.87 × 10⁻⁰⁸ 1.59 × 10⁻⁰⁶ OR0.70 1.92 1.95 2.042 2.105 95% CI 0.56-0.89 1.49-2.48 1.51-2.521.57-2.63 1.56-2.85 Exon 7/9 A307A/ Exon 10 IVS10 Exon 13 Exon 18 Y402HA473A rs203674 Q672Q D936E Iowa # Controls 131 68 129 67 # Cases 404 221404 223 X2 49.4 35.14 0.21 0.64 P 2.09 × 10⁻¹² 3.07 × 10⁻⁰⁹ 0.65 0.8 OR2.82 3.42 1.12 0.89 95% CI 2.11-3.78 2.27-5.15 0.76-1.64 0.51-1.56Columbia # Controls 272 264 264 265 264 # Cases 549 542 545 545 536 X254.4 66.1 66.1 2.05 0.53 P 1.64 × 10⁻¹³ 1.60 × 10⁻¹¹ 4.29 × 10⁻¹⁶ 0.150.46 OR 2.25 2.10 2.44 1.24 1.12 95% CI 1.79-2.75 1.69-2.61 1.97-3.030.937-1.65  0.846-1.49  The frequency of allele 1 and allele 2 from eachSNP was compared between cases and controls and the Yates Chi squared(X2) and P values were calculated along with the Odds Ratio (OR) and 95%confidence interval (95% CI). The actual counts of each genotype aregiven in Tables 6A-6C.

TABLE 5 SSCP, DHPLC and Sequencing Primers SEQ ID SEQ ID Exon RegionForward Primer (5′-3′) NO: Reverse Primer (5′-3′) NO: 1 5′upstream-GCAAAAGTTTCTGATAGGC 64 AATCTTACCTTCTGCTACAC 65 int1 2 int-int2TTAGATAGACCTGTGACTG 66 TCAGGCATAATTGCTAC 67 3 int2-int3 ACTTGTTCCCCCACTC68 CCTCTTTTCGTATGGACTAC 69 int2-ex3 TTGTTCCCCCACTCCTAC 70ACACATTTCCTCCTGTAAGG 71 ex3-int3 CCCTGTGGACATCCTGG 72AACCTCTTTTCGTATGGACTAC 73 4 int3-ex4 ATGCTGTTCATTTTCC 74CCATCCATCTGTGTCAC 75 ex4-int4 ATTACCGTGAATGTGAC 76 TTGTATGAGAAAAAAAAAC77 5 int4-int5 TCCAATCTTATCCTGAGG 78 TCTTACCCACACACTTTG 79 6 int5-ex6GTCCTGGTCACAGTCC 80 GCATACAGCATCTCCTC 81 ex6-int6 GCACTGAATCTGGATG 82ATGAACCTTGAACACAG 83 7 int6-ex7 CGGATACTTATTTCTGC 84 CGTGATTTCATCTCCAG85 ex7-int7 AGAACTGGAGATGAAATC 86 TGAATGGAACTTACAGG 87 8 int7-ex8GTGAAACCTTGTGATTATC 88 TCCCAGTAACTTCCTG 89 ex8-int8 CTGTGATGAACATTTTGAG90 TGCTCTCCTTTCTTCG 91 9 int8-int9 CATTGTTATGGTCCTTAGG 92ACATGCTAGGATTTCAGAG 93 10 int9-ex9 CTTTTTCTTATTCTCTTCCC 94TCACCATCTGCTGTTAC 95 ex9-int10 TGTAACAGCAGATGGTG 96 CCCACAAAAAGACTAAAG97 11 int10-ex11 GGGAAATACTCAGATTG 98 ATGGCATTCATAGTCC 99 ex11-ex11CCAGAACTAAAAATGACTTC 100 GGTAAATCAGACCAACC 101 ex11-int11ATAGTGTGTGGTTACAATG 102 GTTTATGTCAAATCAGGAG 103 12 int11-int12CAAGAAAGAGAATGCGAAC 104 AGATTACAGGCAATGGG 105 13 int12-ex13TTGATTGTTTAGGATGC 106 TTGAGGAGTTCAGGAGGTGG 107 ex13-int13GTGAACTCCTCAATGG 108 ATTACCAATACACACTGG 109 14 int13-int14TTACATAGTGGAGGAGAG 110 TGGAAATGTTGAGGC 111 15 int14-int15AGTTGGTTTGATTCCTATC 112 TTGAGCAGTTCACTTCTG 113 16 int15-ex16TTATGCCCACCTCCAC 114 ATACACTACTGACCAACAC 115 ex16-int16GTCTATGAGAATACAAGCC 116 GAATCTGAGGTGGAGG 117 17 int16-ex17CCCTTTGATTTTCATTC 118 AGAACTCCATTTTCCC 119 ex17-int17 CACAACCACCTCAGATAG120 GCCTAACCTTCACACTG 121 18 int17-ex18 GTCATAGTAGCTCCTGTATTG 122ACGTAACTTCTTCTCCATAC 123 ex18-int18 CTTCCTTGTAAATCTCCAC 124CAATGCACCATACTTATGC 125 19 int18-ex19 TAAAGATTTGCGGAAC 126GGCTCCATCCATTTTG 127 ex19-int19 TTACAAAATGGATGGAG 128AAGTGCTGGGATTACAGGCG 129 20 int19-ex20 CTACTCAAAATGAACACTAGG 130TTTAACCCTGCTATACTCC 131 ex20-int20 TAAATGGAAACTGGACG 132ACCCTATTACTTGTGTTCTG 133 21 int20-int21 GTGTTTGCGTTTGCC 134GAGATTTTTCCAGCCAC 135 22 int21-ex22 TCTCACACTTGCGAAC 136ACCGTTAGTTTTCCAGG 137 ex22- GGTTTGGATAGTGTTTTGAG 138 ATGTTGTTCGCAATGTG139 3′downstream

TABLE 6 Promoter Promoter rs3753394 Intron 1 IVS1 rs529825 Cohort 1 Sumfreq allele 1 freq allele 2 AMD association Chi square Count Allele 1Count Allele 2 Yates χ²/P value OR/95% CI Cohort 2 CON All AMD CON AllAMD 11 CC 126 291 11 GG 149 392 12 CT 114 225 12 GA 95 140 22 TT 24 3322 AA 22 15 Sum 264 549 266 547 freq allele 1 T 0.31 0.25 G 0.74 0.84freq allele 2 C 0.69 0.75 A 0.26 0.16 0 0.095 26.1 2.18E−06 Count allele1 366 807 393 924 Count allele 2 162 291 139 170 Yates χ²/P value 2.840.089 25.4 4.65918E−07 OR/95% CI 1.23 0.977-1.52 1.922 1.49-2.48 Exon 2I62V rs800292 Intron 2 IVS2 insTT Cohort 1 CON AMD CON AMD GG 44 190 SS78 310 GA 18 34 SL 38 73 AA 6 4 LL 10 7 Sum 68 228 126 390 freq allele 1G 0.78 0.91 S 0.77 0.89 freq allele 2 A 0.22 0.09 L 0.23 0.11 AMDassociation X2 P X2 P Chi square 16.19 5.73703E−05 22.19 2.4667E−06 2.792.38 Count Allele 1 Allele 1 106 414 194 693 Count Allele 2 Allele 2 3042 58 87 Yates χ²/P value 15 0.000107511 22.21 2.44398E−06 OR/95% CI2.79 1.67-4.65 2.38 1.65-3.44 Cohort 2 CON All AMD CON All AMD GG 148395 SS 160 409 GA 90 135 SL 95 133 AA 23 16 LL 18 7 Sum 261 546 273 549freq allele 1 G 0.74 0.85 S 0.76 0.87 freq allele 2 A 0.26 0.15 L 0.240.13 29.18756 4.59201E−07 Count allele 1 386 925 415 951 Count allele 2136 167 131 147 Yates χ²/P value 26.12 3.20843E−07 28.4 9.86653E−08OR/95% CI 1.95 1.51-2.52 2.042 1.57-2.63 A307A/ rs1061147/ Intron 6 IVS6rs376644 Exon 7/9 Y402H rs1061170 Cohort 1 CON AMD AA/CC 16 146 AC/CT 56183 CC/TT 59 75 Sum 131 404 freq allele 1 A/C 0.34 0.59 freq allele 2C/T 0.66 0.41 AMD association X2 P Chi square 50.4 1.2593E−12 2.82 CountAllele 1 88 475 Count Allele 2 174 333 Yates χ²/P value 49.4 2.08746E−12OR/95% CI 2.82 2.11-3.78 Cohort 2 CON All AMD 11 TT 186 452 12 CT 76 8922 CC 9 5 Sum 271 546 freq allele 1 T 0.83 0.91 freq allele 2 C 0.170.09 23.82449569 6.70774E−06 Count allele 1 448 993 Count allele 2 94 99Yates χ²/P value 23.04 1.58666E−06 OR/95% CI 2.105 1.56-2.85 Exon 7A307A rs1061147 Exon 9 Y402H rs1061170 Cohort 1 CON AMD CON AMD AA 16146 CC 16 146 AC 56 183 CT 56 183 CC 59 75 TT 59 74 Sum 131 404 131 403freq allele 1 A 0.34 0.59 C 0.34 0.59 freq allele 2 C 0.66 0.41 T 0.660.41 AMD association X2 P X2 P Chi square 50.4 1.2593E−12 50.41.2593E−12 2.82 2.82 Count Allele 1 88 475 88 475 Count Allele 2 174 333174 333 Yates χ²/P value 49.4 2.08746E−12 49.4 2.08746E−12 OR/95% CI2.82 2.11-3.78 2.82 2.11-3.78 Cohort 2 CON All AMD CON All AMD CC 120114 TT 122 118 AC 109 275 CT 113 271 AA 33 158 CC 37 160 Sum 262 547 272549 freq allele 1 C 0.67 0.46 T 0.66 0.46 freq allele 2 A 0.33 0.54 C0.34 0.54 55.19838 1.03234E−12 Count allele 1 349 503 357 507 Countallele 2 175 591 187 591 Yates χ²/P value 59.6 1.16235E−14 54.41.63563E−13 OR/95% CI 2.34 1.89-2.91 2.25 1.79-2.75 Exon 10 A473Ars2274700 Intron 10 IVS10 rs203674 Cohort 1 CON AMD GG 22 145 GA 30 65AA 16 11 Sum 68 221 freq allele 1 G 0.54 0.80 freq allele 2 A 0.46 0.20AMD association X2 P Chi square 36.5 1.54526E−09 3.42 Count Allele 1 74355 Count Allele 2 62 87 Yates χ²/P value 35.14 3.06833E−09 OR/95% C3.42 2.27-5.15 Cohort 2 CON All AMD CON All AMD GG 77 269 11 TT 118 103GA 131 233 12 GT 112 276 AA 56 40 22 GG 34 166 Sum 264 542 264 545 freqallele 1 G 0.54 0.71 T 0.66 0.44 freq allele 2 A 0.46 0.29 G 0.34 0.5646.2 9.24391E−11 66.97458 2.86191E−15 Count allele 1 285 771 348 482Count allele 2 243 313 180 608 Yates χ²/P value 45.4 1.60634E−11 66.14.28616E−16 OR/95% CI 2.10 1.69-2.61 2.44 1.97-3.03 Exon 13 Q672Qrs3753396 Exon 18 D936E rs1065489 Cohort 1 CON AMD CON AMD AA 92 295 GG51 162 GA 33 101 GT 14 56 GG 4 8 TT 2 5 Sum 129 404 67 223 freq allele 1A 0.84 0.86 G 0.87 0.85 freq allele 2 G 0.16 0.14 T 0.13 0.15 AMDassociation X2 P X2 P Chi square 0.309 0.579 0.155 0.694 1.12 0.893Count Allele 1 217 691 116 380 Count Allele 2 41 117 18 66 Yates χ²/Pvalue 0.21 0.65 0.64 0.8 OR/95% C 1.12 0.76-1.64 0.89  0.51-1.56 Cohort2 CON All AMD CON All AMD GG 9 8 TT 9 10 GA 72 138 TG 69 140 AA 184 399GG 186 386 Sum 265 545 264 536 freq allele 1 G 0.17 0.14 T 0.16 0.15freq allele 2 A 0.83 0.86 G 0.84 0.85 2.27 0.322 0.653 0.722 Countallele 1 90 154 87 160 Count allele 2 440 936 441 912 Yates χ²/P value2.05 0.15 0.53 0.46 OR/95% CI 1.24 0.937-1.65 1.12 0.846-1.49

TABLE 7 Frequency of the At-risk Allele in Various Ethnic Groups with orwithout AMD Columbia Iowa African Columbia European Iowa RapanuiControls Hispanic Controls American Cases American Cases MPGN II Risk0.20 0.35 0.35 0.36 0.47 0.55 0.57 0.60 0.69 Haplotype N 52 272 24 13149 549 56 404 20 The frequency of the at-risk haplotype was estimated insamples from different populations from genotypes of the Y402H variantand/or the IVS10 locus. These include Rapanui natives over the age of 65(AMD is extremely rare, and most likely absent, in this Easter Islandpopulation), controls (>65 years of age) from Columbia University,Hispanics general population, controls (>65 years of age) from theUniversity of Iowa, African Americans general population, AMD cases fromColumbia University, European Americans general population, AMD casesfrom the University of Iowa and individuals with MPGNII. N = number ofindividuals.

TABLE 8 Factor H Diplotypes I62V IVS2-18 Y402H D936E IVS20 Risk GG SS CCGG TT Protective AA LL TT GG CC AA LL CT GG CC Protective AA LL TT GG CCAA LL TT GT TT AA LL TT GT CC AA LS CT GG CC AA SS CC GG TT GA LS CT GGTT GA LS CT GG CT GA LS CT GG CC Protective GA LS CT GG CC GA LS CT GTCT GA LS TT GG CT GA LS TT GG CC GA LS TT GG TT GA LS TT GT CC GA LS TTTT CC GA SS CT GG CC GG SS CT GT CC GG SS TT GT TT GG SS TT TT CT GG LLTT GG TT Risk GG SS CC GG TT Risk GG SS CT GG CT GG SS CT GG CC GG SS CTGG CC GG SS CT GT TT GG SS CT GT CT GG SS CT GT CC GG SS CT GT CC GG SSCT GT CC GG SS CT TT CT GG SS TT GG TT GG SS TT GG CT GG SS TT GT TT GGSS TT GT CT GG SS TT GT GT GG SS TT GT CC GG SS TT TT CT GG SS TT TT CCGG SS CC GG TT GG SS CT GG TT GG SS CT GT CC G, A, T, C refer tonucleotides at the indicated polymorphisms. S, L refer to the short andlong (insertion of 2 T nuoleotides) alleles of the intron 2polymorphism.

TABLE 9 Primers Used to Amplify the Factor H Coding Sequence SEQ ID SEQID Exon Forward NO: Reverse NO: 1 TGGGAGTGCAGTGAGAATTG 140GCTAATGATGCTTTTCACAGGA 141 2 CCTGTGACTGTCTAGGCATTTT 142TATGCCTGAATTATATCACTATTGCC 143 3 GCTTTGCTATGTTTAATTTTCCTT 144AACTATGATGGAAATAATTAAATCTGG 145 4 TGCATATGCTGTTCATTTTC 146GTCTTACATTAAAATATCTTAAAGTCTC 147 5 TTTCCTCCAATCTTATCCTGAG 148CGTTCATTCTAAGGAATATCAGCA 149 6 CCTGATGGAAACAACATTTCTG 150AACAGGGCCAGAAAAGTTCA 151 7 TGTTCATTTTAATGCCATTTTG 152AGTTTTCGAAGTTGCCGAAA 153 8 CCTAGAAACCCTAATGGAATGTG 154TGTTCAAGCAAAGTGACCAAA 155 9 TGAGCAAATTTATGTTTCTCATTT 156ATGTCACCTTGTTTTACCAATGG 157 10 TGAATGCTTATGGTTATCCAGGT 158AAAACCTGCAGGAACAAAGC 159 11 TCTTAGAATGGGAAATACTCAGATTG 160TGGTTTTTCAGAAATTCATTTTCA 161 12 ATGTAAAATTAACTTTGGCAATGA 162TTGCTGAAATAAGAATTAGAACTTTG 163 13 TGAATAAAAGAAGAAAATCTTTCCA 164ATCTAAAACACATACATCATGTTTTCA 165 14 AAAACACATACATCATGTTTTCACAA 166GATATGCCTCAACATTTCCAGTC 167 15 GTTGGTTTGATTCCTATCATTTG 168TTGGAAAAGTAATAGGTATGTGTGTC 169 16 CTATGAGAATACAAGCCAAAAGTTC 170TCTCTTGTGCTTCGTGTAAACAA 171 17 AACCCTTTGATTTTCATTCTTCA 172TCAAAGTGAGGGGAATAATTGA 173 18 AATTTATGAGTTAGTGAAACCTGAAT 174TCTTCATTCAAAGTGTAAGTGGTACC 175 19 ACAAAATGGCTAATATATTTTCTCAAG 176TAATGTGTGGGCCCAGCC 177 20 CAAAATGAACACTAGGTGGAACC 178ATTTTGGGGGAGTATAGCAGG 179 21 CTGTTTGCGTTTGCCTTA 180 TTCACGTGGCTGGAAAAATC181 22 TTGAAAACCTGAAAGTCTATGAAGA 182 TCAATCATAAAGTGCACACCTTT 183

TABLE 10 Primers Used to Amplify the CFHR5 Coding Sequence SEQ ID SEQ IDExon Forward NO: Reverse NO: 1 CAGTCCCATTTCTGATTGTTCCA 184GCTGAGGATAATTTGAAGGGG 185 2 GTGATTCATCGATGTAGCTCTTT 186AATGACCAGAGGAGCGTGGAA 187 3 TGATGTCAGTTTTCAAAGTTTTCC 188ACCACTCTCTCAGTTTTGCTAATTAT 189 4 CACATTAAATTTGTTTCTGCAATGA 190AGAAGTGATGAAACAAGAATTTGA 191 5 CCATTTAAGCATTATTTATGGTTTC 192AAACAGGACAGTTACTATTACTTTGCA 193 6 AAATATTTTCAGAGTAAGCACTCATTT 194TTTATCATTTTGATTGGGATTGT 195 7 TGCAGATATTTTATTGACATAATTGTT 196GTTGATCTTGTTGCTTCTTTACAAGA 197 8 CCATTTTCCTGAAACACTACCC 198TCTGTTGCACTGTACCCCAA 199 9 AATTATTTGAATTTCCAGACACCTT 200TTTTGGACTAATTTCATAGAATAACCC 201 10 CTTAAATGCAATTTCACTATTCTATGA 202TAGCCATTATGTAGCC 203

TABLE 11 CFH SNPs in 22 Patients Segregating with MPGNII (AlleleFrequencies (f1 and f2) and Number of Patients by Genotype are Shown) 1EX2 I62V IVS2 −18insTT EX7 A307A IVS7 −53G > T EX9 Y402H EX10 A473A 2rs800292 rs1061147 rs1061170 rs2274700 3 GG 20  (T)9(T)9 20  CC 3 GG 8CC 9 GG 18  4 GA 2 (T)11(T)9 2 CA 10  GT 10  CT 10  GA 4 5 AA 0(T)11(T)11 0 AA 9 TT 4 TT 3 AA 0 6 f1 .95G .95(T)9 .64A .59G .36Y .90G 7f2 .05A .05(T)11 .36C .41T .64H .10A 8 At-Risk Haplotype G 9 A G C GMPGN2-1 G, G 9, 9 A, C G, T C, T G, G MPGN2-2 G, G 9, 9 C, C T, T T, TG, G MPGN2-7 G, G 9, 9 A, C G, T C, T G, G MPGN2-9 G, G 9, 9 A, C G, TC, T G, G MPGN2-10 G, G 9, 9 A, C G, T C, T G, G MPGN2-11 G, G 9, 9 A, CG, T C, T G, A MPGN2-12 G, G 9, 9 A, A T, T C, C G, G MPGN2-13 G, G 9, 9A, C G, T C, T G, G MPGN2-14 G, G 9, 9 A, C G, T C, T G, G MPGN2-15 G, G9, 9 A, A G, G C, C G, G MPGN2-16 G, G 9, 9 C, C T, T T, T G, A MPGN2-17G, G 9, 9 A, A G, G C, C G, G MPGN2-18 G, G 9, 9 A, C G, T C, T G, GMPGN2-19 G, G 9, 9 A, A G, G C, C G, G MPGN2-20 G, G 9, 9 A, A G, G C, CG, G MPGN2-21 G, A 9, 11 C, C T, T T, T G, A MPGN2-22 G, G 9, 9 A, A G,G C, C G, G MPGN2-23 G, G 9, 9 A, A G, G C, C G, G MPGN2-24 G, G 9, 9 A,A G, G C, C G, G MPGN2-27-02 G, A 9, 11 A, C G, T C, T G, A MPGN2-29 G,G 9, 9 A, A G, G C, C G, G MPGN2-30 G, G 9, 9 A, C G, T C, T G, G 1 EX13Q672Q IVS15 −30C > A EX18 D936E IVS18 −89T > C EX20 N1050Y 2 rs3753396rs1065489 3 AA 13 CC 8 GG 13  TT 19  AA 21  4 AG 9 CA 10  GT 9 TC 3 AT 15 GG 0 AA 4 TT 0 CC 0 TT 0 6 f1 .80A .59C .80G .93T .98A 7 f2 .20G .41A.20T .07C .02T 8 At-Risk Haplotype A C G T A MPGN2-1 G, A C, A G, T T, TA, A MPGN2-2 G, A C, A G, T T, T A, A MPGN2-7 G, A C, A G, T T, T A, AMPGN2-9 G, A C, A G, T T, T A, A MPGN2-10 A, A C, A G, T T, T A, AMPGN2-11 A, A C, A G, G T, C A, T MPGN2-12 A, A C, C G, G T, T A, AMPGN2-13 G, A C, A G, T T, T A, A MPGN2-14 G, A C, A G, T T, T A, AMPGN2-15 A, A C, C G, G T, T A, A MPGN2-16 G, A A, A G, T T, C A, AMPGN2-17 A, A C, C G, G T, T A, A MPGN2-18 G, A C, A G, G T, T A, AMPGN2-19 A, A C, C G, G T, T A, A MPGN2-20 A, A C, C G, G T, T A, AMPGN2-21 G, A A, A G, T T, T A, A MPGN2-22 A, A C, C G, G T, T A, AMPGN2-23 A, A C, C G, G T, T A, A MPGN2-24 A, A C, C G, G T, T A, AMPGN2-27-02 A, A C, A G, G T, T A, A MPGN2-29 A, A A, A G, G T, T A, AMPGN2-30 A, A A, A G, G T, C A, A

TABLE 12 Comparison of Factor H SNP Frequencies in 22 MPGNII PatientsVersus Controls (Allele Frequencies Given as f1 and f2) f1 f2 f1 f2 SNPMPGNII MPGNII Controls Controls P-value Exon 2 42 (G)  2 (A) 202 (G)  60(A) 0.0051 I62V IVS2 42 (short)  2 (long) 194 (short)  68 (long) 0.0018−18insTT Exon 7 16 (C) 28 (A)  88 (A) 174 (C) 0.72 A307A Exon 9 28 (H)16 (Y)  88 (H) 174 (Y) 0.00014 Y402H Exon 10 40 (G)  4 (A)  74 (G)  62(A) 0.000013 A473A Exon 13 35 (A)  9 (G) 217 (A)  41 (G) 0.45 Q672Q Exon18 35 (D)  9 (E) 115 (D)  19 (E) 0.32 D936E

TABLE 13 Coding SNPs Associated with MPGNII and the Related ShortConsensus Repeat (SCR) of Factor H SNP SCR Function of SCR Exon 2 I62V 1Interaction with C3b Exon 9 Y402H 7 Heparin binding Interaction with Creactive protein Exon 10 A473A 8 Interaction with C reactive protein

TABLE 14 CFHR5 SNPs in 22 Patients Segregating with MPGNII (AlleleFrequencies (f1 and f2) and Number of Patients by Genotype are Shown) 1Promoter −249T > C Promoter −20T > C IVS1 75T > A Exon2 P46S IVS2 58C >T 2 rs9427661 rs9427662 rs3748557 rs12097550 rs12097550 3 TT 21  TT 21 TT 16  CC 19  CC 16  4 TC 1 TC 1 TA 5 CT 3 CT 5 5 CC 0 CC 0 AA 1 TT 0 TT1 6 f1 .98T .98T .84T .93P .84C 7 f2 .02C .02C .16A .07S .16T At-Risk 8Haplotype T T T C C MPGN2-02 T, T T, T T, T C, C C, C MPGN2-03 T, T T, TT, T C, C C, C MPGN2-07 T, T T, T T, T C, C C, C MPGN2-09 T, T T, T A, TC, C C, T MPGN2-10 T, T T, T T, T C, C C, C MPGN2-11 T, T T, T A, T C, CC, T MPGN2-12 T, T T, T T, T C, C C, C MPGN2-13 T, T T, T A, T C, T C, TMPGN2-14 T, T T, T A, A C, C T, T MPGN2-15 T, T T, T T, T C, T C, CMPGN2-16 C, T C, T A, T C, C C, T MPGN2-17 T, T T, T T, T C, C C, CMPGN2-18 T, T T, T A, T C, C C, T MPGN2-19 T, T T, T T, T C, C C, CMPGN2-20 T, T T, T T, T C, T C, C MPGN2-21 T, T T, T T, T C, C C, CMPGN2-22 T, T T, T T, T C, C C, C MPGN2-23 T, T T, T T, T C, C C, CMPGN2-24 T, T T, T T, T C, C C, C MPGN2-27-2 T, T T, T T, T C, C C, CMPGN2-29 T, T T, T T, T C, C C, C MPGN2-30 T, T T, T T, T C, C C, C

TABLE 15 Comparison of CFHR5 SNP Frequencies in 22 MPGNII PatientsVersus Controls (Allele Frequencies Given as f1 and f2) f1 f2 f1 f2 SNPMPGN II MPGN II Controls Controls P-value Promoter 43 (T) 1 (C) 178 (G)28 (A) 0.033 −249T > C Promoter 43 (T) 1 (C) 178 (G) 28 (A) 0.033 −20T >C IVS1 37 (T) 7 (A) 161 (A) 41 (C) 0.38 +75T > A Exon 2 41 (P) 3 (S) 205(P)  1 (S) 0.00023 P46S IVS2 37 (C) 7 (T) 158 (C) 28 (T) 0.28 +58C > T

TABLE 16A Probes SEQ ID SEQ ID SNP Name Location Reference Allele NO:Variant Allele NO: Promoter 1 5′-TCTGGGATGTAATAATG-3′ 2045′-TCTGGGATGTAATGATG-3′ 205 5′-GAACATTATTACATCCC-3′ 2065′-GAACATCATTACATCCC-3′ 207 rs3753394 Promoter 4 5′-CAGAGGATATCACCAGC-3′208 5′-CAGAGGATATTACCAGC-3′ 209 5′-AGCAGCTGGTGATATCC-3′ 2105′-AGCAGCTGGTAATATCC-3′ 211 rs529825 Intron 1 5′-TACACAGTACGATAGAC-3′212 5′-TACACAGTACAATAGAC-3′ 213 5′-TAAGTCTATCGTACTGT-3′ 2145′-TAAGTCTATTGTACTGT-3′ 215 rs800292 Exon 2 5′-TCTTGGAAATGTAATAA-3′ 2165′-TCTTGGAAATATAATAA-3′ 217 5′-ACCATTATTACATTTCC-3′ 2185′-ACCATTATTATATTTCC-3′ 219 Intron 2 5′-TTTTTTTTTCGTTTTAG-3′ 2205′-TTTTTTTTTTTCGTTTT-3′ 221 5′-CTTTCTAAAACGAAAAA-3′ 2225′-TTCTAAAACGAAAAAAA-3′ 223 rs3766404 Intron 6 5′-TTTAGGACTCATTTGAA-3′224 5′-TTTAGGACTTATTTGAA-3′ 225 5′-TAACTTCAAATGAGTCC-3′ 2265′-TAACTTCAAATAAGTCC-3′ 227 rs1061147 Exon 7 5′-GAAATACAGCAAAATGC-3′ 2285′-GAAATACAGCCAAATGC-3′ 229 5′-ACTTGTGCATTTTGCTG-3′ 2305′-ACTTGTGCATTTGGCTG-3′ 231 rs1061170 Exon 9 5′-TAATCAAAATTATGGAA-3′ 2325′-TAATCAAAATCATGGAA-3′ 233 5′-TTTCTTCCATAATTTTG-3′ 2345′-TTTCTTCCATGATTTTG-3′ 235 rs2274700 Exon 10 5′-AAGAAAAAGCGAAATAT-3′236 5′-AAGAAAAAGCAAAATAT-3′ 237 5′-TTGATATTTCGCTTTTT-3′ 2385′-TTGATATTTTGCTTTTT-3′ 239 Exon 10A 5′-GGATCAAAGA[-]TGACAA-3′ 2405′-GGATCAAAGA[N]TGACAA-3′ 241 5′-GCCCTTGTCA[-]TCTTTG-3′ 2425′-GCCCTTGTCA[N]TCTTTG-3′ 243 rs203674 Intron 10 5′-TTTATTAGTAGATCTAA-3′244 5′-TTTATTAGTATATCTAA-3′ 245 5′-TTGATTAGATCTACTAA-3 2465′-TTGATTAGATATACTAA-3′ 247 rs3753396 Exon 13 5′-ATAAAATTCAATGTGTT-3′248 5′-ATAAAATTCAGTGTGTT-3′ 249 5′-CCATCAACACATTGAAT-3′ 2505′-CCATCAACACACTGAAT-3′ 251 rs375046 Intron 15 5′-TTTATTATAACATTAAT-3′252 5′-TTTATTATAAAATTAAT-3′ 253 5′-TATAATTAATGTTATAA-3′ 2545′-TATAATTAATTTTATAA-3′ 255 rs1065489 Exon 18 5′-CTCCACCTGAGATTTCT-3′256 5′-CTCCACCTGATATTTCT-3′ 257 5′-CATGAGAAATCTCAGGT-3 2585′-CATGAGAAATATCAGGT-3′ 259 Exon 22 5′-TCTTTCATCACGTTCTC-3′ 2605′-TCTTTCATCATGTTCTC-3′ 261 5′-GTGTGAGAACGTGATGA-3′ 2625′-GTGTGAGAACATGATGA-3′ 263

TABLE 16B Primers SEQ ID SEQ ID SNP Name Location Reference Allele NO:Variant Allele NO: Promoter 1 5′-TTTCTGGGATGTAATA-3′(forward) 2645′-TTTCTGGGATGTAATG-3′(forward) 265 5′-CAAAACACTGAACATT-3′(reverse) 2665′-CAAAACACTGAACATC-3′(reverse) 267 rs3753394 Promoter 45′-AAATCCAGAGGATATC-3′(forward) 268 5′-AAATCCAGAGGATATT-3′(forward) 2695′-AAATCAGCAGCTGGTG-3′(reverse) 270 5′-AAATCAGCAGCTGGTA-3′(reverse) 271rs529825 Intron 1 5′-AAGTTTACACAGTACG-3′(forward) 2725′-AAGTTTACACAGTACA-3′(forward) 273 5′-AATGGGTAAGTCTATC-3′(reverse) 2745′-AATGGGTAAGTCTATT-3′(reverse) 275 rs800292 Exon 25′-AGATCTCTTGGAAATG-3′(forward) 276 5′-AGATCTCTTGGAAATA-3′(forward) 2775′-TGCATACCATTATTAC-3′(reverse) 278 5′-TGCATACCATTATTAT-3′(reverse) 279Intron 2 5′-TCATAACTTTTTTTTT-3′(forward) 2805′-ATAACTTTTTTTTTTT-3′(forward) 281 5′-GGGCCTTTCTAAAACG-3′(reverse) 2825′-GCCTTTCTAAAACGAA-3′(reverse) 283 rs3766404 Intron 65′-AATACATTTAGGACTC-3′(forward) 284 5′-AATACATTTAGGACTT-3′(forward) 2855′-ACACTAACTTCAAATG-3′(reverse) 286 5′-ACACTAACTTCAAATA-3′(reverse) 287rs1061147 Exon 7 5′-CCGGGGAAATACAGCA-3′(forward) 2885′-CCGGGGAAATACAGCC-3′(forward) 289 5′-AGTACTTGTGCATTTT-3′(reverse) 2905′-AGTACTTGTGCATTTG-3′(reverse) 291 rs1061170 Exon 95′-GGATATAATCAAAATT-3′(forward) 292 5′-GGATATAATCAAAATC-3′(forward) 2935′-CAAACTTTCTTCCATA-3′(reverse) 294 5′-CAAACTTTCTTCCATG-3′(reverse) 295rs2274700 Exon 10 5′-CTTAAAAGAAAAAGCG-3′(forward) 2965′-CTTAAAAGAAAAAGCA-3′(forward) 297 5′-TTTGCATTGATATTTC-3′(reverse) 2985′-TTTGCATTGATATTTT-3′(reverse) 299 Exon 10A5′-TGAGTGGATCAAAGA[-]-3′(forward) 300 5′-TGAGTGGATCAAAGA[N]-3′(forward)301 5′-CATTGGCCCTTGTCA[-]-3′(reverse) 3025′-CATTGGCCCTTGTCA[N]-3′(reverse) 303 rs203674 Intron 105′-ACCTATTTATTAGTAG-3′(forward) 304 5′-ACCTATTTATTAGTAT-3′(forward) 3055′-CTTTATTGATTAGATC-3′(reverse) 306 5′-CTTTATTGATTAGATA-3′(reverse) 307rs3753396 Exon 13 5′-ACCTAATAAAATTCAA-3′(forward) 3085′-ACCTAATAAAATTCAG-3′(forward) 309 5′-CTCTCCATCAACACAT-3′(reverse) 3105′-CTCTCCATCAACACAC-3′(reverse) 311 rs375046 Intron 155′-TATTTTTTATTATAAC-3′(forward) 312 5′-TATTTTTTATTATAAA-3′(forward) 3135′-AAAAATATAATTAATG-3′(reverse) 314 5′-AAAAATATAATTAATT-3′(reverse) 315rs1065489 Exon 18 5′-TAAATCTCCACCTGAG-3′(forward) 3165′-TAAATCTCCACCTGAT-3′(forward) 317 5′-AACACCATGAGAAATC-3′(reverse) 3185′-AACACCATGAGAAATA-3′(reverse) 319 Exon 225′-TATCGTCTTTCATCAC-3′(forward) 320 5′-TATCGTCTTTCATCAT-3′(forward) 3215′-GCAATGTGTGAGAACG-3′(reverse) 322 5′-GCAATGTGTGAGAACG-3′(reverse) 323

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Although the present invention has been described in detail withreference to specific embodiments, those of skill in the art willrecognize that modifications and improvements are within the scope andspirit of the invention, as set forth in the claims which follow. Allpublications and patent documents cited herein are incorporated hereinby reference as if each such publication or document was specificallyand individually indicated to be incorporated herein by reference.Citation of publications and patent documents (patents, published patentapplications, and unpublished patent applications) is not intended as anadmission that any such document is pertinent prior art, nor does itconstitute any admission as to the contents or date of the same. Theinvention having now been described by way of written description, thoseof skill in the art will recognize that the invention can be practicedin a variety of embodiments and that the foregoing description is forpurposes of illustration and not limitation of the following claims.

1. A diagnostic method for determining a subject's propensity to developage-related macular degeneration (AMD), comprising detecting thepresence or absence of a variation at a polymorphic site of a geneencoding a regulator of complement (RCA).
 2. The method of claim 1,wherein the gene is Factor H or CFHR5.
 3. The method of claim 2, whereinthe polymorphic site is a biallelic single nucleotide polymorphism inthe Factor H gene.
 4. The method of claim 3, comprising obtaining asample of nucleic acid from the patient, and detecting in the DNA of thepatient the presence or absence of a variation at a polymorphic site inthe Factor H gene. 5-7. (canceled)
 8. The method of claim 2, wherein thepolymorphism is rs800292 (162V).
 9. The method of claim 2, wherein thepolymorphism is rs1061170 (Y402H).
 10. The method of claim 2, whereinthe polymorphism is in exon 22 (R1210C).
 11. The method of claim 4,wherein the detecting step comprises (i) combining a nucleic acid samplefrom the subject with one or more polynucleotide probes capable ofhybridizing selectively to a Factor H gene allele in a reaction and (ii)monitoring the reaction to determine the presence of the Factor H geneallele in the sample. 12-16. (canceled)
 17. The method of claim 13,wherein the detecting step comprises (i) mixing in an immunologicalassay a blood or serum sample from the subject with an antibody reagentspecific for the variant protein allele and (ii) monitoring the assay todetermine specific binding between the antibody reagent and the proteinsample.
 18. The method of claim 2, wherein the polymorphic site is asingle nucleotide polymorphism in the CHFR5 gene selected from the groupconsisting of rs9427661 (−249T>C), rs9427662 (−20T>C) and rs120977550(P64S). 19-21. (canceled)
 22. A method of diagnosing susceptibility toage-related macular degeneration (AMD) in a subject, comprisingscreening for a haplotype, wherein the haplotype is: a) a risk haplotypein the Factor H or CFHR5 gene that is more frequently present inindividuals diagnosed with AMD compared to healthy individuals, or b) aprotective haplotype in the Factor H or CFHR5 gene that is morefrequently present in healthy individuals compared to individualsdiagnosed with AMD or c) a neutral haplotype in the Factor H or CFHR5gene.
 23. The method of claim 22, wherein the haplotype is in the FactorH gene.
 24. The method of claim 23, wherein said screening comprisesdetecting the presence or absence of a variant sequence at any one ormore of rs1061170, rs800292, rs1061147, intron 2 (IVS2 or insTT);rs2274700; rs1061170, exon 10A, and exon 22 (R1210C). 25-28. (canceled)29. A method for prevention or treatment of age-related maculardegeneration (AMD) comprising administering a Factor H polypeptide to asubject in need of such treatment.
 30. The method of claim 29, whereinthe subject has a Factor H haplotype associated with AMD risk.
 31. Themethod of claim 29, wherein the Factor H polypeptide comprisesisoleucine at position
 62. 32-41. (canceled)
 42. A pharmaceuticalcomposition comprising a recombinant Factor H polypeptide, wherein therecombinant Factor H polypeptide comprises isoleucine at position 62,wherein said composition is free of pathogens and suitable foradministration to a human patient. 43-48. (canceled)
 47. An isolatedhost cell expressing a recombinant variant human Factor H, wherein saidvariant has histidine in place of tyrosine at amino acid 402 (Y402H), orthe variant has cysteine in place of arginine at amino acid 1210(R1210C).
 49. A method of screening for an agent capable of treatingAMD, comprising: contacting a host cell expressing a recombinant varianthuman Factor H, wherein said variant has histidine in place of tyrosineat amino acid 402 (Y402H), or the variant has cysteine in place ofarginine at amino acid 1210 (R1210C) with the agent; and monitoringexpression or processing of the variant. 50-51. (canceled)
 52. A deviceor kit for diagnosing susceptibility to AMD in a subject comprisingoligonucleotides that distinguish alleles at least one polymorphic sitein the Factor H gene listed in Table A and/or any one or more ofrs1061170, rs800292, rs1061147, intron 2 (IVS2 or insTT); rs2274700;rs1061170, exon 10A, and exon 22 (R1210C). 53-60. (canceled)